Antibody based gene therapy with tissue-directed expression

ABSTRACT

Embodiments of the disclosure include methods and compositions for treatment of a medical condition related to the liver, including at least viral infections and liver cancer, for example. In specific embodiments, immunotherapies are provided for delivering polynucleotides locally to the liver, wherein the polynucleotides encode particular gene products that include bispecific antibodies, including those that target certain liver antigens, for example.

PRIOR RELATED APPLICATIONS

This application is a national phase application under 35 U.S.C § 371that claims priority to International Application No. PCT/US2017/054570filed Sep. 29, 2017, which claims priority to U.S. Provisional PatentApplication Ser. No. 62/402,504, filed Sep. 30, 2016, and also to U.S.Provisional Patent Application Ser. No. 62/505,955 filed May 14, 2017,all of which are incorporated herein by reference in their entirety.

INCORPORATION BY REFERENCE

The sequence listing file, BLG16-034-USCON.xml, created on Jan. 30,2023, sized 65,908 bytes, submitted on Jan. 30, 2023, is incorporatedherein by reference for all purposes.

FIELD OF THE DISCLOSURE

The present disclosure concerns at least the fields of immunology, cellbiology, molecular biology, and medicine.

BACKGROUND OF THE DISCLOSURE

The field of immunotherapy has promise in treating various infectiousdiseases and cancers with recent success in clinical trials. Throughengineering or activating host immune cells, either artificially invitro or in vivo, these diseases can potentially be reversed. However,these therapies can hold significant toxicities, lack of persistence,and dosing limitations. New methods that solve these limitations,maximizing the efficacy of treatment while minimizing off-target andoff-organ toxicity are needed.

The present disclosure satisfies a long-felt need in the art byproviding effective targeted immunotherapies for medical conditions thataffect a particular tissue or organ, while lacking systemic side effectsand toxicity, in at least certain aspects.

SUMMARY OF THE DISCLOSURE

Embodiments of the disclosure concern methods and/or compositions fortreatment of a disease in a specific tissue. In some embodiments, thedisclosure concerns targeted therapy using compositions that areimmunotherapeutic. In specific embodiments, the immunotherapies areuseful for one or more liver-associated medical conditions.

Some embodiments of the disclosure concern gene therapy using deliveryof a polynucleotide encoding a secretable form of a protein havingmultiple entities (such as a bipartite or tripartite protein, forexample) that can bind substantially at the same time to an antigenspecific for an organ or tissue and can also bind to a target thatindirectly or directly stimulates an immune response. In particularaspects, the multi-component protein is able to bind a liverdisease-specific antigen and to bind a target that facilitatesre-direction of the immune system to diseased cells, for examplelocalized in the liver. In specific embodiments, the composition is abi-specific antibody and may also include a linker to connect operablythe different components. In particular embodiments the compositions ofthe secretable polypeptides are delivered to an individual in needthereof in vivo in nucleic acid or protein form.

In specific embodiments the composition is provided locally and in theform of gene therapy such that constant and high production may occur onsite in the desired tissue or organ, including the liver. That is, oncethe polynucleotide is delivered to a tissue or organ into cells locally,or via systemic injection and organ localization by the delivery vector,or via system injection and selective expression in the organ (all asexamples), the local cells in the tissue are transduced and produce andsecrete the desired protein. In embodiments wherein cancer is beingtreated, this may occur within the solid tumor mass and/or within thetumor microenvironment, for example.

Embodiments of the disclosure include methods of directly delivering Tcell activation into a specific organ in vivo, thereby modulating thelocal immune response. This differs from recombinant protein strategieswhere antigen specificity guides to target organs, or from usingcell-based carriers for delivery.

In certain embodiments of bispecific polypeptides, such as bispecificantibodies, the two parts may or may not function independently of eachother, and in cases where they function independently of one anotherthey may impart a synergistic effect, although in some cases the effectis additive.

In particular embodiments, a mixture of bispecific antibodies coveringtwo or more different non-overlapping epitopes may be utilized toencompass the large majority of serotypes for a given virus (includingglobal HBV serotypes) or variations within a cancer, therebycompensating for different affinities of the antibodies. In addition,introducing multiple antibody genes at the same time into the livercould generate bi-, tri-, quadra- or more specific antibodies through Fcpairing of heterologous genes, resulting in synergistic binding tomultiple epitopes (including multiple HBsAg epitopes, as an example), inat least some cases.

Two different strategies may be utilized for efficient organ (such ashepatic) gene delivery of the bispecific antibodies, although otherstrategies may be employed. The first strategy may utilize a vector suchas an adeno-associated virus (AAV) vector to deliver bispecificantibodies. AAV in specific cases affords a more permanent expression ofbispecific antibodies that protects the individual from de novoinfection cycles, but results herein suggest induced inflammation woulddestabilize the AAV genome quickly, leading to a transient, albeit safetherapy, in at least some cases. Furthermore, in specific embodimentsthe inflammation vaccinates against AAV capsid, preventingre-administration of therapy.

As an alternative approach one could deliver mRNA encoding bispecificantibodies directly into the liver, which would generate expression overtime (starting with hours and lasting several days) that is well withintherapeutic kinetics established herein, and yield a safe strategy withreliable pharmacokinetics. In such cases, the mRNA may be optimized byone or more means to prevent immune activation, increase stability,reduce any tendency to aggregate, such as over time, and/or to avoidimpurities. Such optimization may include the use of modifiednucleosides (for example, with 1-methylpseudouridine) in the mRNA and/ormay include particular 5′ UTRs, 3′UTRs, and/or poly(A) tail for improvedintracellular stability and translational efficiency (see, e.g., Stadleret al., 2017, Nat. Med.).

The present disclosure provides novel methods of giving individuals anadaptive immune system to fight HBV infection. In certain embodiments,this is achieved through a single bispecific antibody molecule, whichprovides humoral immunity via the antibody portion, but also the abilityto link and activate T cells against infected hepatocytes. Use of such acombination encompasses mechanisms utilizing covalently closed circularDNA (cccDNA) degradation, direct killing of infected cells, viral entryinhibition, innate immune activity, HBsAg secretion inhibition, andvaccine-like immune stimulation in a single therapeutic product, in atleast some embodiments.

In one embodiment, there is a composition comprising a polynucleotideencoding a secretable polypeptide that comprises at least one tissueantigen-targeting entity (such as liver antigen or lung antigen, forexample) and at least one immunostimulatory entity. The polypeptide mayfurther comprise a linker region operably linking the at least one liverantigen-targeting entity and the at least one immunostimulatory entity.In specific embodiments, the liver antigen comprises an antigen on aliver cell, for example a diseased liver cell, such as a cancer cell.The cancer cell may be a primary liver cancer cell, such as ahepatocellular carcinoma cell or a hepatoblastoma cell. In specificcases, the cancer cell is derived from a cancer that metastasized to theliver. Regarding the liver antigen, it may be an antigen on a pathogenthat infects the liver, and the pathogen may be a virus, bacteria, orfungus. In specific cases the virus is a Hepatitis A virus, Hepatitis Bvirus, Hepatitis C virus, Hepatitis D virus, or Hepatitis E virus. Insome cases, the virus is Cytomegalovirus, Epstein-Barr virus, JC virus,BK virus, HSV-1, HSV-2, varicella zoster, HHV-6, HHV-8, Ebola virus,Zika virus, parvovirus, severe acute respiratory syndrome(SARS)-associated coronavirus, papillomavirus, influenza virus, orYellow fever virus.

In particular cases, the liver antigen is HBV small surface antigen, HBVmiddle surface antigen (includes PreS2 domain), HBV large surfaceantigen (includes PreS1 and PreS2 domains), HBV core antigen, HBV eantigen, HCV E1 protein, HCV E2 protein, EBV glycoprotein, CMVglycoprotein, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33,EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38,CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA,PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptoralpha, ERBB2 (Her2/neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, EphrinB2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2,Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta,TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialicacid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K,OR51E2, TARP, WTI, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7,MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2,Fos-related antigen 1, p53, p53 mutant, prostein, survivin andtelomerase, PCTA-1/Galectin 8, MelanA/MARTI, Ras mutant, hTERT, sarcomatranslocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17,PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS,SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerasereverse transcriptase, RU1, RU2, intestinal carboxyl esterase, muthsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A,BST2, EMR2, LY75, GPC3, FCRL5, or IGLL1. The liver antigen-targetingentity may comprise a single chain antibody, single chain variablefragment (scFv), peptide, camelid variable domain, shark IgNAR variabledomain, single domain antibody, affimer or VHH antibody.

In certain embodiments, the immunostimulatory entity comprises a singlechain antibody, single chain variable fragment (scFv), peptide, camelidvariable domain, shark IgNAR variable domain, single domain antibody,affimer or VHH antibody against a receptor on an immune cell thatprovokes stimulation. The immunostimulatory entity may also comprise acytokine, Fc receptor-binding entity, an ectodomain of an immune cellligand, or a combination thereof. Specific cytokines includeinterleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13,IL-14, IL-15, IL-16 and IL-18, hematopoietic factors such asgranulocyte-macrophage colony stimulating factor (GM-CSF), granulocytecolony stimulating factor (G-CSF) and erythropoeitin, tumor necrosisfactors (TNF) such as TNFα, lymphokines such as lymphotoxin, interferonssuch as interferon a, interferon f), and interferon y, or variouschemokines, or a combination thereof. In cases wherein an Fereceptor-binding entity is employed, it may be an IgG constant region,such as one from IgG4, IgG1, IgG3, or IgG2. In specific cases, the Fcreceptor-binding entity comprises a monoclonal antibody that binds an Fcreceptor. The Fc receptor-binding entity may comprise an scFv or singledomain antibody that binds an Fc receptor.

In specific embodiments, the immunostimulatory entity comprises ananti-CD3 scFv, an anti-CD28 scFv, anti-41BB scFv, anti-OX40 scFv,anti-CTLA4 scFv, an anti-CD16 scFv, anti-PD1 scFv, anti-PD-L1 scFv,anti-CD47 scFv, part or all of the ectodomain for a ligand for CD28(such as part or all of the ectodomain of CD80 and/or CD86), part or allof the ectodomain of 41BB ligand, SIRPalpha, part or all of theectodomain of the LIGHT protein, ICOS-ligand, CD276 (B7-H3), B7-H4, andB7-H6, CD134L, or CD137L, and/or a combination thereof.

Regarding the inker, the linker may comprise a glycine-serine sequence,an Fe domain, one or more immunoglobulin domains, pairing ofheterologous antibody light and heavy chain constant domains, or acombination thereof. In specific cases, the Fe domain comprises thehuman IgG1, IgG2, IgG3, or IgG4 Fe domains. The Fe domain may compriseone or more mutations that alters a property of the domain. The Fedomain may comprise a mutation that reduces FcRy receptor binding,reduces the ability of the Fe domain to have complement binding, reducesthe ability of the Fe domain to form immune complexes, and/or rendersthe domain to be monomeric in structure. In specific embodiments, theimmunoglobulin domain is configured as a spacer for antigen binding. Theimmunoglobulin domain may comprise an immunoglobulin domain selectedfrom the group consisting of extracellular regions of human proteinsCD80, CD86, CDS, CD22, CD19, CD28, CD79, CD278, CD7, CD2, LILR, KIR, andCD4. In certain cases, the linker comprises one or more CH2 and/or CH3domain(s) from one or more antibodies, which may containing mutationsfor monomeric forms in some embodiments. The linker may comprise theFeRn binding domain.

In particular embodiments, the secretable polypeptide comprises thefollowing structure in a N-terminal to C-terminal orientation: liverantigen-targeting entity-linker-immunostimulatory entity; orimmunostimulatory entity-linker-liver antigen-targeting entity. Thepolypeptide may comprise the following structure in a N-terminal toC-terminal orientation: liver antigen-targeting entity-linker-cytokine;or cytokine-linker-liver antigen-targeting entity.

In certain aspects polynucleotides may comprise RNA or DNA, and they mayor may not be comprised in or on a vector or delivery vehicle, such as aviral vector (adenoviral vector, an adeno-associated viral vector, aretroviral vector, herpes virus vector, baculovirus vector or alentiviral vector) or a non-viral vector or delivery vehicle, such as alipid-based nanoparticle, a polymeric-based nanoparticle, or an exosome.In certain cases, when the polynucleotide is a messenger RNA (mRNA), themRNA may or may not comprise modified nucleotides. In other cases, whenthe polynucleotide is a messenger RNA (mRNA), the mRNA comprisesunmodified nucleotides. The mRNA may comprise one or more modifiednucleotides. The vector or delivery vehicle may comprise an expressioncassette that encodes the polypeptide. The expression cassette maycomprise one or more regulatory sequences, such as sequences thatcomprise at least one tissue-specific regulatory sequence, including aliver-specific regulatory sequence. In specific cases, thetissue-specific regulatory sequence comprises a thyroxine bindingglobulin (TBG) promoter, a regulatory element as described in US2011/0184049, albumin enhancer/promoter, apoE promoter, alpha1-antitrypsin promoter, or HBV core promoter (as examples). In caseswherein the vector is an adeno-associated viral vector, it may comprisean adeno-associated virus comprising a mutated capsid or is a serotypethat transduces human liver. The adeno-associated viral vector may beAAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or an AAV serotype isolated from a non-human primate. In some cases,the vector or delivery vehicle comprises a moiety that directs deliveryof the vector or delivery vehicle to the liver.

In specific embodiments, the polynucleotide that encodes the secretablepolypeptide also encodes a cytoprotective agent, although in certaincases they are comprised on separate polynucleotides. A cytoprotectiveagent may be delivered to an individual as a nucleic acid or as apolypeptide. The cytoprotective agent may be an apoptosis inhibitor. Inspecific embodiments, the cytoprotective agent is Bcl2, Bel-XL, Mcl-,CED-0, Bfl-1, X-linked inhibitor of apoptosis protein (XIAP), c-IAP1,C-IAP2, NAIP, Livin, Survivin, serpin proteinase inhibitor 9, orSERPINB4, and other gene products that inhibit apoptosis. In specificcases, the cytoprotective agent is an siRNA, shRNA, miRNA, antisenseoligonucleotide, or a morpholino that targets Fas receptor, TNF alphareceptor, Bax, Bid, Bak, or Bad and other gene products that otherwisepromote apoptosis. In specific cases, the cytoprotective agent is a mRNAthat comprises untranslated sequences that are targetable by an miRNAmolecule of the individual restricting expression in desired cell typesin a target tissue.

In one embodiment, there is a method of treating a medical condition,comprising the step of delivering to an individual with or at risk forthe medical condition a therapeutically effective amount of at least oneof the compositions encompassed by the disclosure (polynucleotide orpolypeptide, for example). In specific embodiments, the medicalcondition is cancer or an infectious disease, such as Hepatitis B orHepatitis C infection. The composition may be delivered to theindividual more than once, and in such cases the duration betweenseparate deliveries of the composition is within days, weeks, or monthsof one another. In some cases, a mixture of compositions is provided tothe individual. The individual may be receiving, has received, or willreceive an additional treatment for the medical condition, such asPaclitaxel, Doxorubicin, 5-fluorouracil, Everolimus, Melphalan,Pamidronate, Anastrozole, Exemestane, Nelarabine, Belinostat,Carmustine, Bleomycin, Bosutinib, Irinotecan, Vandetanib, Bicalutamide,Lomustine, Clofarabine, Cabozantinib, Dactinomycin, Cobimetinib,Cytoxan, Cyclophosphamide, Decitabine, Daunorubicin, Cytarabine,Docetaxel, Hydroxyurea, Decarbazine, Leuprolide, epirubicin,oxaliplatin, Asparaginase, Estramustine, Vismodegib, Amifostine,Flutamide, Toremifene, Panobinostat, Fulvestrant, Letrozole, Degarelix,Fludarabine, Pralatrexate, floxuridine, Gemcitabine, Afatinib, ImatinibMesylate, Carmustine, Eribulin, Altretamine, Topotecan, Hydrea(Hydroxyurea, Palbociclib, Ponatinib, Idarubicin, Ifosfamide, Ibrutinib,Axitinib, Gefitinib, Romidepsin, Ixabepilone, Ruxolitinib, Cabazitaxel,Carfilzomib, Lenvatinib, Chlorambucil, Sargramostim, Cladribine,Trifluridine and Tipiracil, Leuprolide, Olaparib, Mitotane,Procarbazine, Megestrol, Trametinib, Mesna, Strontium-89 Chloride,Methotrexate, Mechlorethamine, Mitomycin, Vinorelbine, Sorafenib,nilutamide, Pentostatin, Mitoxantrone, Sonidegib, Alitretinoin,Carboplatin, Cisplatin, Pomalidomide, Mercaptopurine, Zoledronic acid,Lenalidomide, Octreotide, Tamoxifen, Dasatinib, Regorafenib, Histrelin,Sunitinib, Omacetaxine, Thioguanine, Dabrafenib, Erlotinib, Bexarotene,Decarbazine, Paclitaxel, Docetaxel, Temozolomide, Thiotepa, Thalidomide,Temsirolimus, Bendamustine hydrochloride, Triptorelin, Arsenic trioxide,lapatinib, Valrubicin, Histrelin, Vinblastine, Bortezomib, Etoposide,Tretinoin, Azacitidine, Vincristine, Pazopanib, Teniposide, Leucovorin,Crizotinib, Capecitabine, Enzalutamide, Trabectedin, Streptozocin,Vemurafenib, Goserelin, Vorinostat, Zoledronic acid, Everolimus,Idelalisib, Ceritinib, Abiraterone, or a combination thereof. One ormore HBV antivirals may be provided to the individual. The compositionmay be delivered locally or systemically. In specific cases, thecomposition is delivered by injection intravenously, by directedinjection using catheters into the portal vein or into hepatic artery,orally administered, subcutaneously injected, intramuscularly injected,or intraperitoneal injected. The delivery may or may not be by constantinfusion.

In one embodiment, there is a polypeptide comprising the followingcomponents: 1) an antibody or antibody fragment comprising XTL19 scFv,XTL17 scFv, OST577 scFv, 5a 19 scFv, or a combination thereof; 2) IgG1wildtype Fc, IgG4 wiltype Fc, IgG1(AA) Fc, IgG2(AA) Fc, IgG1(AA)-CH2domain only, IgG2(AA)-CH2 domain only, IgG4m Fc, IgG4m, CD80 ectodomain,CD86 ectodomain, or a combination thereof; and 3) anti-CD3 scFv, whereinin a N-terminal to C-terminal orientation the components of 1), 2), or3) may be in any order. In certain cases, in a N-terminal to C-terminalorientation the order is 1), 2) and 3). In other cases, in a N-terminalto C-terminal orientation the order is 3), 2), and 1).

In another embodiment there is a method of treating a medical condition,comprising the step of delivering to an individual with or at risk forthe medical condition a therapeutically effective amount of two separatepolypeptides or one or more polynucleotides encoding the two separatepolypeptides, wherein a first of the polypeptides comprises a liverantigen-targeting entity operably linked to a FcRn binding domain and asecond of the polypeptides comprises a liver antigen-targeting entityoperably linked to anti-CD3 scFv, such that the first polypeptide caninhibit the secretion of antigen particles, while the second polypeptidecan not be inhibited by said particles and instead redirects T cells toa pathogenic cell's surface. In specific embodiments, the liverantigen-targeting entity operably linked to FcRn binding domain inhibitsthe secretion of surface antigen particles and Hepatitis B virions froma liver cell. In specific aspects, the second polypeptide is abispecific antibody that activates T cells for proliferation,cytotoxicity, and cytokine release in the presence of the liver antigen.In one embodiment, the second polypeptide remains on a liver cellsurface without internalization into the liver cell, thereby prolongingengagement with effector cells.

In one embodiment, provided herein is a method of generatingmonospecific and bispecific antibodies in situ in tissue (liver or lung,as examples) of an individual, comprising the step of providing to theindividual two or more polynucleotides that each encode non-identicalmonospecific antibody polypeptides, wherein the antibodies produced fromthe polynucleotides in situ in tissue of the individual dimerize to eachother, thereby generating a mixture of monospecific antibodies andbispecific antibodies within the tissue of the individual. The antibodymay comprise one or more antigen binding domains that comprise a singlechain antibody, single chain variable fragment (scFv), peptide, camelidvariable domain, shark IgNAR variable domain, single domain antibody,affimer or VHH antibody. In some cases, the antigen-binding domainsdimerize in their respective Fc regions, and in some cases theantigen-binding domains dimerize with a separate protein domain (such asone that comprises leucine zipper motifs, hinge and CH2 domain fromimmunoglobulin G, helix-loop-helix dimerization domain, or proteindomain forming disulfide bonds). In some embodiments, at least one ofthe polynucleotides encodes a monospecific antibody for a diseaseantigen and at least one of the polynucleotides encodes a monospecificantibody for an immunostimulatory agent or serves an immunostimulatoryagent domain. The immunostimulatory agent may be an anti-CD3 scFv, ananti-CD28 scFv, anti-41BB scFv, anti-OX40 scFv, anti-CTLA4 scFv, ananti-CD 16 scFv, anti-PD1 scFv, anti-PD-L1 scFv, anti-CD47 scFv, part orall of the ectodomain for a ligand for CD28, part or all of theectodomain of 4 IBB ligand, SIRPalpha, part or all of the ectodomain ofthe LIGHT protein, ICOS-ligand, CD276 (B7-H3), B7-H4, and B7-H6, CD134L,or CD137L, and/or a combination thereof. In some cases, part or all ofthe ectodomain for a ligand for CD28 is further defined as part or allof the ectodomain of CD80 and/or CD86.

In certain embodiments, there are methods of generating bispecific,trispecific and quadraspecific antibodies in situ in tissue (forexample, liver or lung) of an individual, comprising the step ofproviding to the individual two or more polynucleotides that each encodenon-identical bispecific antibody polypeptides, wherein the antibodiesproduced from the polynucleotides in situ in the tissue of theindividual dimerize to each other, thereby generating a mixture ofbispecific, trispecific and quadraspecific antibodies within the tissueof the individual. The antibody may comprise one or more antigen bindingdomains that comprise a single chain antibody, single chain variablefragment (scFv), peptide, camelid variable domain, shark IgNAR variabledomain, single domain antibody, affimer or VHH antibody. The antibodiesmay dimerize in their respective Fc regions. The antigen-binding domainsmay dimerize with a separate protein domain, such as leucine zippermotifs, hinge and CH2 domain from immunoglobulin G, helix-loop-helixdimerization domain, or protein domain forming disulfide bonds. Inspecific embodiments, at least one of the polynucleotides encodes anantibody for a disease antigen and at least one of the polynucleotidesencodes a monospecific antibody for an immunostimulatory agent or servesan immunostimulatory agent domain. In certain cases, theimmunostimulatory agent is an anti-CD3 scFv, an anti-CD28 scFv,anti-41BB scFv, anti-OX40 scFv, anti-CTLA4 scFv, an anti-CD 16 scFv,anti-PD1 scFv, anti-PD-L1 scFv, anti-CD47 scFv, part or all of theectodomain for a ligand for CD28, part or all of the ectodomain of 4 IBBligand, SIRPalpha, part or all of the ectodomain of the LIGHT protein,ICOS-ligand, CD276 (B7-H3), B7-H4, and B7-H6, CD134L, or CD137L, and/ora combination thereof. Part or all of an ectodomain for a ligand forCD28 may be further defined as part or all of the ectodomain of CD80and/or CD86.

In some embodiments, there are compositions that when the secretablepolypeptide is expressed at suitable levels in a tissue in vivo, thepolypeptide elicits antigen-independent properties of immunostimulationin addition to antigen-dependent immunostimulation toward the antigen.In some embodiments, there is a composition comprising animmunostimulatory monospecific antibody without an antigen-targetingdomain that comprises activity of signaling and activating immune cellswhen expressed in tissue in vivo, but that lacks the same activity invitro.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention. The scope of the present application is notintended to be limited to the particular embodiments of the process,machine, manufacture, composition of matter, means, methods and stepsdescribed in the specification.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawings, in which:

FIG. 1 illustrates an example of a bispecific antibody of thedisclosure;

FIG. 2 illustrates an example of a mechanism of action for a particularbispecific antibody of the disclosure, without being bound by theory;

FIG. 3 exemplifies a particular plasmid for use in a model for HepatitisB infection;

FIG. 4 demonstrates in vivo efficacy of one particular bispecificantibody encompassed by the disclosure;

FIG. 5 shows examples of in vivo dosing for one particular bispecificantibody encompassed by the disclosure;

FIG. 6 demonstrates in vivo efficacy utilizing two examples of doses ina mouse model;

FIGS. 7A-7E. Antibody production was validated in vivo and a viralbioluminescence system for immune readout was established. (7 A) The19-Fc antibody was developed consisting of an scFv-Fc fusion proteinderived from the 19.79.5 human antibody clone with specificity to the“a” determinant of HBsAg. The Fc domain was derived from the human IgG1protein. Antibody was cloned into a pCAGGS vector for expression invivo. (7B) Upon hydrodynamic injection, serum collected four days postinjection and measured for functional 19-Fc protein that could bindHBsAg (n=4). (7C) A luminesce model was developed for HBV, combiningdelivering a wildtype HBV overlength genome with using the HBV corepromoter to drive GFP and firefly luciferase expression. (7D) IVISimaging revealed efficient and specific luminescence specific to theliver after injection of 5 ug of plasmid. (7E) Luminescence wasmonitored overtime in NSG mice, revealing a gradual decline inexpression over 2.5 months (n=4, unpaired t-test, *=p<0.05);

FIGS. 8A-8B. Bispecific antibody formats were screened for efficacy invivo. (8A) Various bispecific antibody formats depicted were cloned intopCAGGS vectors, with the immune versus HBsAg binding components appendedto different ends with different linker domains inserted. (8B) 15 ug ofbispecific antibody vector was co-injected with 5 ug HBV-Luc, andluminescence measured at Day 4 post injection (n=3) (unpaired t-test,*=p<0.05). Definitions: mCD3=scFv against murine CD3epsilon derived from2c11 hamster clone; mB7.1=ectodomain of mouse B7.1 protein;CH2,CH3=components of antibody Fc domain, gray=human IgG1, brown=humanIgG4 with mutations to prevent Fc receptor binding, orange=human IgG1with mutations to prevent dimerization and Fc receptor binding;XTL19=scFv derived from antibody clone 19.79.5;

FIGS. 9A-9C. Bispecific antibodies reduce virus in an acute HBV mousemodel. (9 A) A dosing series was undertaken in order to find the plasmidlevel yielding the highest decrease in luminescence compared to Gaussiacontrol at Day 4 post hydrodynamic injection with 5 ug HBV-Luc (n=4,unpaired t-test, *=p<0.05). (9B) Using the 15 ug dose, 19-Fc-mCD3 wascompared to 19-Fc lacking mCD3 binding, as well as version containingG4m lacking Fc receptor binding. Co-injection of Gaussia and EmptypCAGGS served as controls (n=4, unpaired t-test, *=p<0.05). Backgroundluminescence in the assay was 5×10⁵ p/sec/cm². (9C) T the decrease inluminescence was correlated by monitoring serum HBsAg levels in theexperiment, which normalized to the Day 4 HBsAg level of theHBV-Luc+Gaussia condition (n=4, unpaired t-test, *=p<0.05);

FIG. 10A-10D. Bispecific antibodies binding CD3 yield target antigenindependent T cell activation. (10A) The XTL19 specificity was replacedwith 139 scFv specificity to EGFRvIII and scFv against human EphA2, withsimilar groups to the previous test (15 ug Ab+5 ug HBV-Luc) (n=4,unpaired t-test, *=p<0.05). (10B) 19-G4m-mCD3 has significantly moreluminescence reduction than 139-G4m-mCD3 at day 4 post injection,revealing a role for antigen targeting. (n=4, unpaired t-test). (IOC)Bispecific antibodies were constructed to lack an Fc domain similar tothe BiTE format, along with mCD3 scFv alone (n=4, unpaired t-test,*=p<0.05). (10D) The kinetics of treatment response for the BiTE formatswas followed over 16 days;

FIGS. 11A-11D. Expression length, safety, and clearance mechanism wasevaluated for bispecific antibody therapy. (11 A) Contribution ofadaptive immunity in the context of bispecific antibody therapy wasassessed by comparing 19-G4m-mCD3 therapy in NSG versus immunocompetentmice, showing reduction stopped in NSG mice after initial bispecificmediated reduction. (1 IB) The time course of initial bispecificantibody activation was followed over the first 4 days, with significantdecrease in luminescence occurring at Day 1, and not increasingthereafter (unpaired t-test). Q IC) Toxicity of bispecific antibodyexpression was assessed co-injected Cre plasmid with 19-G4m-mCD3 versusGaussia into Rosa-Luc mice. Differences in luminescence were notsignificant, indicating hepatocyte survival (n=4, unpaired t-test,*=p<0.05). (11D) CRISPR-Cas9 therapy was compared against 19-Fc-mCD3 inthe same system, with similar significant reductions in luminescence,albeit with different mechanisms of action (n=4, unpaired t-test,*=p<0.05);

FIGS. 12A-12D. Bispecific antibodies can promotes HBV cccDNA clearanceand host immune response in a novel mouse model of HBV. (12A) ACre/LoxP-HBV (CLX) plasmid was used to generate cccDNA in vivo, whilealso marking infected hepatocytes with luciferase expression in Rosa-Lucmice. 5 ug CLX plasmid was co-injected with 15 ug 19- or 139-G4m-mCD3plasmid or 15 ug CMV-Gaussia, and luminescence levels monitored.Infected hepatocytes appeared to be cleared in the bispecific treatedgroups, as judged by drops in luminescence. (12B) The Day 4 luminescencelevels among the different groups was compared to assess any initialcytotoxicity against infected cells. (n=4, unpaired t-test, *=p<0.05).(12C) HBsAg serum levels were also assessed at Day 4 post injection,with the bispecific constructs yielding over 1.5 log reduction in serumHBsAg. (n=4, unpaired t-test, *=p<0.05, **=p<0.001) (12D) The ability ofbispecific antibodies to trigger adaptive immunity was tracked measuringthe serum levels of anti-HBsAg antibodies, with differences starting atday 12 (n=4, unpaired t-test, *=p<0.05);

FIG. 13 . Sequence Information for the HBV-Luc plasmid (SEQ ID NO: 1 andSEQ ID NO:2). A reported system for monitoring the immune responseagainst HBV in realtime using bioluminescence was developed. The plasmidpSP65-HBVayw1.3 (gift of Stefan Wieland) was adapted for use. Theendogenous HBV core promoter was utilized, in order to accurately assessthe effects of cytokines on HBV gene expression. To utilize nativeelements, GFP was introduced as a fusion protein shortly after coreprotein translation, simplifying construction. Luciferase generated hadno foreign sequences, being separated by 2A peptide cleavage;

FIG. 14 . Dual monitoring system for infected hepatocytes usingbioluminescence and cccDNA generation. Cre/LoxP-HBV plasmid contains aCMV-NLS-Cre(intron) cassette and a LoxP-HBV flanked genome, with theLoxP inserted between amino acid 83 and 84 of the HBV X protein. Plasmidwas hydrodynamically injected into Rosa-Luc mice, which contain drivenby the Rosa26 promoter a LoxP-STOP-LoxP-firefly luciferase cassette.Upon introduction, CMV driven Cre recombinase expression will bothexcise and form a recombinant cccDNA molecule, and activate luciferaseexpression in the same cell. Thus, every cell that has HBV cccDNA willalso have luciferase expression, affording dual monitoring and readouts;

FIG. 15 . Testing if therapeutic efficacy of delivering immunestimulating molecules against HBV would be maintained when cells alsoexpressed an apoptotic inhibitor to prevent cell death. Mice (n=4+ wereall hydrodynamically injected with 5 ug HBV-Luc plasmid, along with 5 ugCAG-Bcl2 or Control plasmid, plus 15 ug 19-Fc-mCD3 plasmid. Expressingan anti-apoptotic protein inside cells does not inhibit bispecificantibody efficacy as judged by equivalent bioluminescence decrease tocontrol condition;

FIGS. 16A-16B. Brodynamic tail vein injection of pCAG.HBs-Fc results inHBs-Fc antibody expression in vivo. (16A) Scheme of pCAG.HBs-Fc. (16B)Serum was collected 4 days post hydrodynamic tail vein injection ofpCAG.HBs-Fc. The serum concentration of HBs-Fc was determined by HBsAgIgG Ab ELISA (n=4, * p<0.05);

FIGS. 17A-17B. HBs-Fc gene delivery into hepatocytes has anti-HBV invivo. (17A) Immunocompetent mice were co-injected by hydrodynamic tailvein injection with 5 μg pUBVffLuc and 15 μg pCAG.HBs-Fc, pCAG.EvIII-Fc,or control plasmid. Quantitative bioluminescence imaging data(radiance=photons/sec/cm²/sr) for all mice are shown (mean±s.e.m, n=3, *p<0.05). (17B) HBsAg levels were determined by ELISA. Data wasnormalized to the day 4 HBsAg level of the pUBV-ffLuc/control plasmidgroup (mean±s.e.m., n=3, * p<0.05);

FIGS. 18A-18C. Addition of CD3 binding domain enhances anti-HBV activityof HBs-Fc. (18A) Scheme of antibody constructs (green: HBsAg-specificscFv derived from mAb 19.79.5 (19), purple: EvIII-specific scFv derivedfrom mAb 139 (139), gray: IgG1-Fc (CH2, CH3), blue: murine CD3-specificscFv derived from mAb 145-2C11 (145). (18B) Immunocompetent mice wereco-injected by hydrodynamic tail vein injection with 15 μgpCAG.HBs-Fc-CD3, pCAG.EvIIIFc-CD3, pCAG.HBs-Fc, pCAG-EvIII-Fc or controlplasmid and 5 μg pHBV-ffLuc. Quantitative bioluminescence imaging data(radiance=photons/sec/cm²/sr) for all mice are shown (mean±s.e.m, n=3).(18C) Mouse images for different constructs are shown for day 4 postinjection (n=3, * p<0.05) using the identical exposure time;

FIGS. 19A-19B. Fc receptor binding does not contribute to the anti-HBVactivity of HBs-Fc-CD3. (19A) Scheme of antibody constructs (green:HBsAg-specific scFv derived from mAb 19.79.5 (19), purple:EvIII-specific scFv derived from mAb 139 (139), brown: IgG4-Fc withmutated Fc receptor binding sites (CH2, CH3), blue: murine CD3-specificscFv derived from mAb 145-2C11 (145). (19B) Immunocompetent mice wereco-injected by hydrodynamic tail vein injection with 15 μg ofpCAG.HBs-mFc-CD3, pCAG.EvIII-mFc-CD3, or control plasmid with 5 μgpHBV-ffLuc. Quantitative bioluminescence imaging data(radiance=photons/sec/cm²/sr) for all mice are shown (mean±s.e.m, n=3).pCAG.HBs-mFc-CD3 had significantly greater anti-HBV activity thanp.CAG-EvIII-mFc-CD3 (* p<0.05);

FIGS. 20A-20E. In vivo expression of HBs-mFc-CD3 in a recombinant cccDNAmodel of HBV has antiviral effects and induces endogenous HBsAgantibodies. Rosa-Luc mice were co-injected by hydrodynamic tail veininjection with 5 μg pCLX and 15 μg pCAG.HBs-mFc-CD3, pCAG.EvIII-mFc-CD3,or 15 μg control plasmid (n=4). (20A) HBsAg serum levels were determinedby ELISA at day 4 post injection (mean±s.e.m is shown, n=4, ** p<0.005,*** p<0.0001). (20B) The development of host HBsAg IgG Abs wasdetermined by ELISA at the indicated time points (mean±s.e.m is shown,n=4, * p<0.05). (20C) Tissue was harvested from mice at day 4 postinjection, and HBV core protein (red) expression was assessed byimmunofluorescence (blue=DAPI, scale bar=50μιη). (20D,20E) Quantitativebioluminescence imaging data (radiance=photons/sec/cm²/sr) for all miceis shown on (20D) day 4 post injection, and (20E) during long-termfollow up (mean±s.e.m, * p<0.05);

FIGS. 21A-21C. Murine model that allows for measuring the clearance ofHBV using non-invasive bioluminescence imaging. (21 A) Scheme of plasmidencoding the overlength (1.3-mer) HBV genome and a core protein fusedGFP-2A-ffLuc cassette, both under the transcriptional control ofidentical HBV core promoters. (21B,21C) NSG mice were injected with 5 μgof pHBV-ffLuc by hydrodynamic tail vein injection, and ffLuc expressionwas monitored by bioluminescence imaging. (2 IB) Bioluminescence imagesof mice on day 4 post injection. (21C) Quantitative bioluminescenceimaging data (radiance=photons/sec/cm²/sr) is shown over time (mean andstandard error mean, n=4);

FIG. 22 . In vivo expression of EphA2-Fc-CD3 had anti-HBV activity. Toconfirm the antigen-independent activity of EvIII-Fc-CD3, the inventorsreplaced the EvIII-specific scFv is pCAG.EvIII-Fc-CD3 with a scFvderived from the EphA2-specific mAb 4H5 pCAG.EphA2-Fc-CD3).Immunocompetent mice were co-injected by hydrodynamic tail veininjection with 5 μg pHBV-ffLuc and 15 μg pCAG.EvIII-Fc-CD3,pCAG.Epha2-Fc-CD3, or control plasmid. Quantitative bioluminescenceimaging data (radiance=photons/sec/cm²/sr) for all mice are shown forday 4 post injection (mean±s.e.m,), n=4, n.s.=not significant, **p<0.005);

FIG. 23 . The antiviral effects of in vivo expression of bispecificantibodies are consistent across multiple experiments. Collatedbioluminescence data (radiance=photons/sec/cm²/sr) of replicates acrossthe different experiments for each construct are depicted demonstratingconsistent effects. Control, n=16; pCAG.EvIII-Fc, n=6;pCAG.EvIII-Fc-CD3, n=4; pCAG.EvIII-mFc-CD3, n=5; pCAGHBs-Fc, n=6;pCAG.HBs-Fc-CD3, n=10; pCAG.HBsmFc-CD3, n=9;

FIGS. 24A-24B. Bispecific antibodies act early after injection andthrough CD3 engagement to mediate antiviral activity. (24A)Bioluminescence was followed after co-injection 15 μg pCAG.HBs-mFc-CD3or Control and 5 μg pHBV-ffLuc over the first 4 days postinjection inmice (n=3). (24B) In a similar experiment, 15 μg pCAG.HBs-mFc-CD3, 15 μgpCAG.CD80-mFc-HBs, or Control and 5 μg pHBV-ffLuc were injected intomice and measured at day 4 post-injection (n=4). Quantitativebioluminescence imaging data (radiance=photons/sec/cm²/sr) for all miceare shown (mean±s.e.m,) and significant differences denoted (*p<0.05,*** p<0.0001);

FIGS. 25A-25C. In vivo expression of HBs-mFc-CD3 in hepatocytes isnontoxic. (25 A) Transaminase levels (AST and ALT) were measured at day4 post-injection of 5 pHBVffLuc and 15 μg pCAG.HBs-Fc, pCAG.HBs-mFc-CD3,control plasmid (n=3), or pCAG.EvIIImFc-CD3 (n=4). (mean±s.e.m.). Therewas no significant (n.s.) difference between any of the groups in eitherALT or AST measurements. (25B) Toxicity of HBs-mFc-CD3 expression wasassessed by co-injecting pCMV-LS-Cre with pCAG.HBs-mFc-CD3 or controlplasmid into Rosa-Luc mice containing a reporter LoxP-STOP-LoxP-ffluccassette inducing ffLuc expression in transduced, Crerecombinase-expressing hepatocytes. Quantitative bioluminescence imagingdata (radiance=photons/sec/cm²/sr) for all mice are shown (mean±s.e.m.,n=3). There was no significant (n.s.) difference betweenpCAG.HBs-mFc-CD3 and control plasmid injected groups. (25 C) Livertissue of mice was harvested at day 4 post injection in mice co-injectedwith 5 μg pHBV-ffLuc and 15 μg pCAG.HBs-mFc-CD3 or control plasmid,fixed in paraformaldehyde, and tissue stained with hematoxylin andeosin. No difference in tissue morphology was observed between mice (Lowmagnification scale bar=100μπι, High magnification scale bar=50μπι);

FIG. 26 . In vivo expression of HBs-Fc, HBs-Fc-CD3, or EvIII-Fc-CD3 inhepatocytes is nontoxic. Liver tissue was harvested at day 4 posthydrodynamic tail vein injection of mice co-injected with 5 μg pHBV-Lucand 15 μg pCAG.HBs-Fc-CD3, pCAG.HBs-Fc, pCAG.EvIII-Fc-CD3, or controlplasmid. Tissues were fixed in paraformaldehyde, and sections werestained with hematoxylin and eosin (Low magnification scale bar=100μπι,High magnification scale bar=50μπι);

FIG. 27A-27B. Recombinant cccDNA HBV mouse model to monitor antiviralactivity and hepatoxicity of antiviral agents. (A) Scheme of pCLX, whichcontains a CMVNLS-Cre (intron) cassette and a LoxP-HBV flanked genome(derived from pLoxP-HBV), with the LoxP site inserted between amino acid83 and 84 of the HBV X protein. When pCLX is injected by hydrodynamictail vein injection into Rosa-Luc mice, which contain aLoxP-STOPLoxP-ffLuc cassette driven by the Rosa26 promoter, Crerecombinase expression will i) excise and form a recombinant (r)cccDNAmolecule, and ii) induce ffLuc expression in the same cell. Thus, everycell that contains HBV rcccDNA will also express ffLuc enabling toxicitymonitoring of antiviral agents by non-invasive bioluminescence imaging.(B) 20 μg pCLX or pLoxP-HBV was injected by hydrodynamic tail veininjection into NSG mice. Serum was collected one week post injection andHBsAg levels were measured by ELISA (mean±s.e.m., n=4). (C) 5 μg pCLX orpLoxP-HBV were injected by hydrodynamic tail vein injection into miceand 4 days post injection liver sections were stained for HBV core(red=HBV core, blue=DAPI, scale bar=20μιη); and

FIGS. 28A-28B. Sequence information for pHBV-ffLuc. (28A) (SEQ ID NO: 1)Core protein sequence (blue) fused to GFP reading frame (green) toenable downstream expression of GFP-2A-ffLuc. (28B) (SEQ ID NO:2) DNAsequence of core-GFP fusion, with the transcriptional start site forcore mRNA indicated in red (same as the canonical pregenomic pgRNA), thestart codon for the core protein is indicated in blue, and the startcodon of GFP is indicated in green.

DETAILED DESCRIPTION

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more. In specificembodiments, aspects of the invention may “consist essentially of or“consist of one or more sequences of the invention, for example. Someembodiments of the invention may consist of or consist essentially ofone or more elements, method steps, and/or methods of the invention. Itis contemplated that any method or composition described herein can beimplemented with respect to any other method or composition describedherein. The scope of the present application is not intended to belimited to the particular embodiments of the process, machine,manufacture, composition of matter, means, methods and steps describedin the specification.

In particular cases, the present disclosure concerns a strategy oftargeting viral infections and cancers of the liver using, for example,bispecific antibodies that re-direct the immune system toward thediseased cells. Previously in the art, such molecules have beendeveloped as recombinant proteins administered to an individual in needthereof. In clinical trials targeting various tumors, the efficacy ofthis strategy has been poor and limited to only a few successesrequiring constant infusion (for example, blinatumomab targeting CD 19+cells). In the present disclosure, polynucleotides encoding theseproteins may be delivered directly to the affected organ, such that theywill be highly concentrated and reach the diseased cells readily, asopposed to systemic therapies. Furthermore, this reduces thedose-limited toxicities noted in the previous trials for this class ofmolecule.

The present disclosure particularly concerns applications targeting theliver tissue, which is the host of various infectious diseases andcancer disorders (both primary and metastatic). Toward targeting theliver, gene therapy embodiments can utilize adeno-associated virus, orlipid-nanoparticle (LNP) mRNA delivery, for example, both of which havealready been validated for efficacy in humans and chimpanzeesrespectively. Specifically, encompassed herein are bispecific antibodiesagainst Hepatitis B virus that have resulted in a 100-fold decrease inviral genomes within 4 days of treatment compared to an untreated groupin immunocompetent mice, and there is clearance of the virus by 8 days,compared to viral genomes being unaffected at that point in untreatedmice. Similar efficacy has been observed in monospecific constructstargeting T cell activation alone. Considering that current therapiescannot target the viral genome, this is a significant advance in theart. Encompassed herein are examples of different antibody sequences andbispecific designs that yield efficacy in mice at least. Alsoencompassed herein are unique linker compositions that can maintainproper geometry for efficacy and T cell activation, for example. Thepresent disclosure demonstrates the ability to target an autologousantigen in the liver, as an example of a specific organ, and theclearance of signal in those cells, which may be extrapolated forapplication into a number of autologous tumor antigen targets, forexample.

I. Examples of Compositions

The present disclosure provides novel compositions for use in thetreatment or prevention of one or more symptoms of a medical condition,such as one that affects a specific organ, such as the liver, which willbe utilized as an example hereafter. In specific embodiments, thecompositions comprise one or more components that are able to bind aliver disease antigen, which includes an antigen on a cell that islocated in the liver or an antigen on a cell outside the liver but thatis also found on a cell that is located in the liver. In certainembodiments the compositions comprise one or more components that areable to stimulate and/or activate the immune system, including stimulateand/or activate the immune system to focus on the liver, includingdiseased cells in the liver. The composition(s) may also comprise atleast one linker that operably links two or more components of themolecule. The two or more components may work in conjunction with oneanother or may work separately from one another. The two or morecomponents of the composition(s) may have similar or separate functionsand/or they may each be able to bind different targets. Upon binding oftheir respective targets, they may directly or indirectly result indownstream action(s) that may or may not be separate downstream actions.

A. Liver Disease Antigen-Targeting Entity

Embodiments of the disclosure include compositions that comprise atleast one liver disease antigen-targeting entity. The entity maycomprise 1, 2, 3, 4, or more liver antigen-targeting entities. Whenmultiple liver disease antigen-targeting entities are present on onemolecule of the composition, the different entities may abut oneanother, or there may be sequence in between them on the molecule thatis not of a liver antigen-targeting entity. In cases wherein there issequence between two liver disease antigen-targeting entities, the liverantigen-targeting entities are still configured such that they mayfunction properly, including acting in conjunction, in at least certaincases. The different liver disease antigen-targeting entities may or maynot have the same liver antigen to target, and certain embodimentsencompass a cocktail of liver disease antigen-targeting entities,towards one more antigens, being administered to an individual at thesame time.

In particular embodiments, the liver antigen-targeting entity targets anantigen on the surface of a cell in the liver. That cell may be a normalliver cell or a diseased liver cell. In specific embodiments, the liverantigen-targeting entity is able to directly or indirectly bind at leastone antigen on a cell in the liver. In cases wherein the cell in theliver is a diseased cell, the cell may infected with a pathogen, such asa virus or bacteria or parasite. In cases wherein the pathogen is aparasite, the targeted antigen may be from Plasmodium falciparum, thecausative agent of malaria. Other parasites infecting the liver thatcould be targeted include Plasmodium vivax, Plasmodium ovale, Plasmodiummalariae, Plasmodium knowlesi, Toxoplasmosis gondii, Trypanosoma cruzi,Echinococcosis, Fasciola hepatica, Clonorchis sinensis, Schistosomajaponicum, Schistosoma mansoni, Schistosoma intercalatum, Ascarislumbricoides, Baylisascaris procyonis, Toxocara canis, or Toxocara cati.In cases wherein the pathogen is a virus, the liver antigen-targetingentity may target a cell that expresses an antigen from any type ofHepatitis virus, such as Hepatitis A, Hepatitis B, Hepatitis C,Hepatitis D, or Hepatitis E virus, or another type of virus, such asCytomegalovirus, Epstein-Barr virus, JC virus, BK virus, HSV-1, HSV-2,varicella zoster, HHV-6, HHV-8, Ebola virus, Zika virus, parvovirus,severe acute respiratory syndrome (SARS)-associated coronavirus,papillomavirus, influenza virus, or Yellow fever virus. In other cases,the liver disease antigen-targeting entity targets an antigen on acancer cell that is in the liver (such as hepatocellular carcinoma orhepatoblastoma), including a primary cancer cell, a cancer cell thatoriginates from a cancer that has metastasized to the liver, arefractory cancer cell, and so forth.

The liver antigen-targeting entity may be of any kind, so long as it isable to bind directly or indirectly to the liver antigen. The liverantigen-targeting entity may be a protein or peptide, including thatencoded by a particular polynucleotide that may be provided to anindividual in need thereof (although in alternative embodiments thecomposition provided to the individual is a peptide or a polypeptide andnot a polynucleotide). In particular embodiments, the liverantigen-targeting entity comprises an antibody or functional fragmentthereof, including a single chain antibody, a single chain variablefragment, a single domain antibody, a camelid antibody, or a llamaantibody, for example. Other examples include affimers. Specificexamples of liver antigen-targeting entities includes those that targetat least HBV small surface antigen, HBV middle surface antigen (includesPreS2 domain), HBV large surface antigen (includes PreS1 and PreS2domains), HBV core antigen, HBV e antigen, HCV E1 protein, HCV E2protein, EBV glycoprotein, CMV glycoprotein, and so forth. For cancerswithin the liver including metastases, specific examples of antigensthat could be targeted include TSHR, CD 19, CD123, CD22, CD30, CD171,CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3,FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin,IL-IRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20,Folate receptor alpha, ERBB2 (Her2/neu), MUC1, EGFR, NCAM, Prostase,PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl,tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2,Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97,CD 179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1,ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a,MAGE-A1, legumain, HPV E6,E7, MAGE A1, ETV6-AML, sperm protein 17,XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53mutant, prostein, survivin and telomerase, PCTA-1/Galectin 8,MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoints,ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor,Cyclin B1, MYCN, RhoC, TRP-2, CYPIB I, BORIS, SART3, PAX5, OY-TES1, LCK,AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2,intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1,FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, andIGLL1, for example.

In certain cases, the liver antigen-targeting entity comprises one ormore antibodies or antibody fragments, such as an scFv. Particular scFvsmay include those that directly bind at least one Hepatitis viralantigen, such as scFvs derived from monoclonal antibodies 17.1.41,19.79.5, OST577, scFv A5, VHH-S4, VHH-S5, HzKR127, KR359, 2B6, 2D9, 2E7,2G3, ADRI-2F3, E6F6, HB-C7A, 5alpha19, and 1C9, for example.

In certain cases, the liver antigen-targeting entity comprises one ormore peptides or peptide fragments. Particular peptides may includethose that directly bind at least one Hepatitis viral antigen.Particular peptides may include those that directly bind at least oneHepatitis viral antigen, such as Peptide A5, Peptide ETGAKPH, PeptideP7, Peptide pC, Peptide p2, Peptide p5, Peptide pi 8, Peptide 4B10, orPeptide SRLLYGW, for example.

In certain cases, the liver antigen-targeting entity comprises twoscFvs, such as tandem scFvs. In particular, the tandem scFvs binddifferent liver antigen-targeting entities, although in some cases theliver antigen-targeting comprises tandem scFvs that bind the sameantigen, for example at the same or different site on the antigen; theymay or may not bind different epitopes on the same antigen.

In specific embodiments, a liver antigen-targeting entity is operablylinked, such as on a fusion protein, to a component that binds one ormore immunoglobulin receptors, such as an Fc receptor. The liverantigen-targeting entity may be linked to an FcRn binding domain, forexample. Compositions wherein the liver antigen-targeting entity islinked to one or more FcRn binding domains provides activity ofinhibition of secretion of surface antigen particles and HBV virions (asan example) from a liver cell. The Fc domain may comprise a mutationthat reduces FcRy receptor binding and cytotoxicity, reduces the abilityof the Fc domain to inhibit complement binding, reduces the ability ofthe Fc domain to form immune complexes, and/or renders the domain to bemonomelic in structure.

In another case, the linker region between the liver diseaseantigen-targeting entity and a CD3 binding domain will lack FcRnbinding, such as via mutations in an Fc domain linking the two moieties,preventing the hepatocyte from endocytosis of the antibody therapeutic.Mutations at residues Ile253Ala, Ser254Ala, His435Ala and Tyr436Alaencompassing residues at CH2-CH3 interface of human IgG Fc domains serveto abrogate FcRn binding and can be used in this embodiment. Such afeature may be desirable in order to maximize the ability of immunecells to target liver disease antigens on the cell surface via retainingappropriate efficacious geometries in treating various diseases, whilealso optimizing more of the bispecific molecule on the surface of thetarget cell as opposed to intracellular trafficking.

Single chain antibody sequences that may be utilized in specificembodiments are as follows:

XTL19 (targets HBsAg) (SEQ ID NO: 3)QVQLVESGGG VVQPGGSLRL SCAPSGFVFR SYGMHWVRQT PGKGLEWVSLIWHDGSNRFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAMYFCARERLIAAPAAF DLWGQGTLVT VSSGGGGSGG GGSGGGGSSY VLTQPPSVSVAPGKTARISC GGNNIGTKNV HWYQQKPGQA PVLVVYADSD RPSGIPERFSGSNSGNTATL TISRVEVGDE ADYYCQVWDS VSYHVVFGGG TTLTVLGXTL 17 (targets HBsAg) (SEQ ID NO: 4)QVQLVESGGG VVRPGRSLRL SCAASGFAFS DYSINWVRQA PGKGLEWVAIISYDGRITYY RDSVKGRFTI SRDDSKNTLY LQMNSLRTED YDFWSGSSVGRNYDGMDVWG LGTTVTVSSG GGGSGGGGSG GGGSDIVMTQ TAVYYCARQYSPLSLSVTPG EPASISCRSS QSLLHRSGNN YLDWYLQKPG HSPQLLIYVGSNRASGVPDR FSGSGSGTEY TLRISTVEAE DVGVYYCMQA LQTPRTFGQG TKLEIKRQST577 (targets HBsAg) (SEQ ID NO: 5)QVQLVESGGG VVQPGRSLRL SCAASGFTFS RYGMHWVR QAPGKGLEWVAVISYDGSNK WYADSVKGRF TISRDNSKNT LFLQMHSL RAADTGVYYCAKDQLYFGSQ SPGHYWVQGT LVTVSSGGGG SGGGGSGGGG SQSQLTQPPSVSVAPGQTAR ITCGGDNIGS KSVNWFQQKP GQAPVLVVYD DNERPSGISERFSGSNSGNT ATLTISRVEA GDEADYYCQV WDSSSDHVVF GGGTKLTVLHul2F6 (targets human CD3 epsilon) (SEQ ID NO: 25)DIQMTQSPSS LSASVGDRVT MTCRASSDSV SYMHWYQQTP GKAPKPWIYATSNLASGVPS RFSGSGSGTD YTLTISSLQP EDIATYYCQQ WSSNPPTFGQGTKLQITRGG GGSGGGGSGG GGSQVQLVQS GGGVVQPGRS LRLSCKASGYTFTSYAMYWV RQAPGKGLEW VAIINPSSGY TKNQKFDRFT ISADKSKSTAFLQMDSLRPE DTGVYFCARD GDYDVYFSAS CFGPDYWGQG TPVTVSSHumanized UCHT1 (targets human CD3 epsilon) (SEQ ID NO: 26)DIQMTQSPSS LSASVGDRVT ITCRASQDIR NYLNWYQQKP GKAPKLLIYYTSRLESGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQQ GNTLPWTFGQGTKVEIKRTG GGGSGGGGSG GGGSEVGQLV ESGGGLVQPG GSLRLSCAASGYSFTGYTMN WVRQAPGKGL EWVALINPYK GVTTYADSVK GRFTISVDKSKNTAYLQMNS LRAEDTAVYY CARSGYYGDS DWYFDVWGQG TLVTVSSHuM291 (targets human CD3 epsilon) (SEQ ID NO: 27)QVQLVQSGAE VKKPGASVKV SCKASGYTFI SYTMHWVRQA PGQGLEWMGYINPRSGYTHY NQKLKDKATL TADKSASTAY MELSSLRSED TAVYYCARSA YYDYDGFAYW GQGTLVTVSS GGGGSGGGGS GGGGSDIQMT QSPSSLSASVGDRVTITCSA SSSVSYMNWY QQKPGKAPKR LIYDTSKLAS GVPSRFSGSGSGTDFTLTIS SLQPEDFATY YCQQWSSNPP TFGGGTKVEI KgQKT3-5 (targets human CD3 epsilon) (SEQ ID NO: 28)QVQLVQSGGG VVQPGRSLRL SCKASGYTFT RYTMHWVRQA PGKGLEWIGYINPSRGYTNY NQKVKDRFTI STDKSKSTAF LQMDSLRPED TAVYYCARYY DDHYCLDYWG QGTPVTVSSG GGGSGGGGSG GGGSDIQMTQ SPSSLSASVG DRVTITCSAS SSVSYMNWYQ QTPGKAPKRW IYDTSKLASG VPSRFSGSGS GTDYTFTISS LQPEDIATYY CQQWSSNPFT FGQGTKLQIT R gQKT3-7 (targets human CD3 epsilon) (SEQ ID NO: 29)QVQLVQSGGG VVQPGRSLRL SCKASGYTFT RYTMHWVRQA PGKGLEWIGY INPSRGYTNY NQKVKDRFTI SRDNSKNTAF LQMDSLRPED TGVYFCARYY DDHYCLDYWG QGTPVTVSSG GGGSGGGGSG GGGSDIQMTQ SPSSLSASVG DRVTITCSAS SSVSYMNWYQ QTPGKAPKRW IYDTSKLASG VPSRFSGSGS GTDYTFTISS LQPEDIATYY CQQWSSNPFT FGQGTKLQIT RTGN1412 (targets human CD28) (SEQ ID NO: 30)QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYYIHWVRQA PGQGLEWIGCIYPGNVNTNY NEKFKDRATL TVDTSISTAY MELSRLRSDD TAVYFCTRSHYGLDWNFDVW GQGTTVTVSS GGGGSGGGGS GGGGSDIQMT QSPSSLSASVGDRVTITCHA SQNIYVWLNW YQQKPGKAPK LLIYKASNLH TGVPSRFSGSGSGTDFTLTI SSLQPEDFAT YYCQQGQTYP YTFGGGTKVE IK

Additional single chain variable fragments that can be employed intissue-directed bispecific antibody expression are provided below:

-   -   scFv A5 (against HBsAg, Reference PMID: 14597165)    -   VHH-S4 (against HBsAg, Reference PMID: 19085971)    -   VHH-S5 (against HBsAg, Reference PMID: 19085971)    -   HzKR127 (against PreS1, Reference PMID: 18176536)    -   KR359 (against PreS1, Reference PMID: 10772975)    -   2B6 (against PreS1, Reference PMID: 26888694)    -   2D9 (against PreS1, Reference PMID: 26888694)    -   2E7 (against PreS1, Reference PMID: 26888694)    -   2G3 (against PreS1, Reference PMID: 26888694)    -   ADRI-2F3 (against HBsAg, Reference PMID: 25923526)    -   E6F6 (against HBsAg, Reference PMID: 26423112)    -   HB-C7A (against HBsAg, Reference PMID: 18479762)    -   5a19 (against PreS1, Reference PMID: 11749974)    -   scFv 1C9 (against HBV core protein, PMID: 10385671

Peptides can also be used as the targeting ligand toward HBsAg, alone orin conjunction with other moieties. In some embodiments, the peptideswill form a dual binding agent, wherein a peptide and scFv againstdifferent HBsAg epitopes or proteins are joined in tandem to result inbivalent binding to target antigen. Particular embodiments would utilizea PreS1 binding peptide in conjunction with an antibody that binds tothe small HBsAg protein. Regardless of the embodiment, some contemplatedpeptides targeting HBV envelope for use in chimeric antigen receptorsmay utilize the sequence listed below:

Peptide A5 (targets PreSI, Reference PMID: 24966187): (SEQ ID NO: 6)SGSGLKKKWST Peptide ETGAKPH (targets HBsAg, Reference PMID: 16087122):(SEQ ID NO: 7) CETGAKPHC Peptide P7(targets PreSI, Reference PMID: 21856287): (SEQ ID NO: 8) KHMHWHPPALNTPeptide pC (targets PreS1, Reference PMID: 17192308): (SEQ ID NO: 9)SGSGWTNWWST Peptide p2 (targets PreS1, Reference PMID: 17192308):(SEQ ID NO: 10) NNWWYWWDTLVN Peptide p5(targets PreSI, Reference PMID: 17192308): (SEQ ID NO: 11) GLWRFWFGDFLTPeptide pi 8 (targets PreSI, Reference PMID: 17192308): (SEQ ID NO: 12)WTDMF T AW W STP Peptide 4B 10(targets PreSI, Reference PMID: 27384014): (SEQ ID NO: 13)LRNIRLRNIRLRNIRLRNIR Peptide SRLLYGW(targets PreSI, Reference PMID: 15996026): (SEQ ID NO: 14) CSRLLYGWC

Examples of scFvs that may be employed to target a cancer antigen are asfollows:

-   -   scFv 4H5 (against human EphA2, PMID: 17241664)    -   scFv 3E11 or 2G9 or 4G5 or 3D8 or 2E10 (against human        glypican-3, PMID: 22564378)    -   scFv hulG8 (against human prostate stem cell antigen, PMID:        19010866)    -   scFv HMFG2 (against human MUC1, PMID: 18354214)    -   scFv 139 (against human EGFRvIII, PMID: 22780919)    -   scFv P4 (against human mesothelin, WO2013063419 A2)    -   scFv C2-45 (against human carcinoembryonic antigen, PMID:        20683006)    -   scFv C4 (against human folate receptor-alpha, PMID: 26101914)

B. Immunostimulatory Entity

Embodiments of the disclosure include compositions that comprise one ormore immunostimulatory entities. The composition may comprise 1, 2, 3,4, or more immunostimulatory entities. When multiple immunostimulatoryentities are present on a molecule of the composition, the differententities may abut one another, or there may be sequence in between themon the molecule that is not of an immunostimulatory entity. In caseswherein there is sequence on the molecule between two immunostimulatoryentities, the immunostimulatory entities are still configured such thatthey may function properly, including acting in conjunction, in at leastcertain cases.

In particular embodiments, one or more immunostimulatory entities in thecomposition directs the immune system of an individual to which thecomposition is provided toward certain cells in the body, includingdiseased liver cells in the body. The composition(s) elicit T cellactivation and cytokine secretion, in at least some embodiments. Inparticular embodiments, the immunostimulatory domain(s) activate Tcells, NK cells, NK T cells, macrophages, monocytes, basophils,neutrophils, eosinophils, mast cells, Kupffer cells, or B cells at theliver and indirectly or directly cause activation of signaling pathwaysin immune effector cells. The composition(s) may facilitate recruitmentof T cells to the surface of cells in the liver, including diseasedcells in the liver, such as cancer cells or pathogen-infected cells inthe liver. In some embodiments, the protein secreted will just containone or more immunostimulatory domains with or without linkers, and lacka liver disease antigen targeting domain. This serves to stimulate theimmune response within the organ, without any particular redirection ofimmune cells towards a diseased cell. The mechanism may includeaggregation of secreted protein made in tissue to provide stimulation tosurrounding cells.

The immunostimulatory entity may comprise a polypeptide or a peptide, ora polynucleotide encoding a polypeptide or peptide. In specific cases,the immunostimulatory domain comprises an antibody or functionalantibody fragment, including an scFv. Particular examples include ananti-CD3 scFv, an anti-CD28 scFv, anti-41BB scFv, anti-OX40 scFv,anti-CTLA4 scFv, an anti-CD 16 scFv, anti-PD1 scFv, anti-PD-L1 scFv,anti-CD47 scFv, part or all of the ectodomain for a ligand for CD28(such as part or all of the ectodomain of CD80 and/or CD86), part or allof the ectodomain of 4 IBB ligand, SIRPalpha, part or all of theectodomain of the LIGHT protein, ICOS-ligand, CD276 (B7-H3), B7-H4, andB7-H6, CD134L, CD137L, a cytokine, or a combination thereof, forexample. Examples of cytokines include interleukin-2 (TL-2), IL-4, IL-5,IL-6, IL-7, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16 and IL-18,hematopoietic factors such as granulocyte-macrophage colony stimulatingfactor (GM-CSF), granulocyte colony stimulating factor (G-CSF) anderythropoeitin, tumor necrosis factors (TNF) such as T Fa, lymphokinessuch as lymphotoxin, interferons such as interferon a, interferon β, andinterferon γ, and chemokines, and a combination thereof.

In some embodiments, the polynucleotide delivered will encode an immunestimulating protein without any antigen-binding component. For theseembodiments, localization to the target organ may be achieved by thespecificity of tissue delivery alone. Furthermore, the entity will onlyactivate immune cells and not form synapses between the diseased celland immune cells. This offers the ability to stimulate immune cellsalone, or in certain cases, provide therapy for diseases that do notexpress disease specific-antigen targets on the cell surface. Theactivation of immune cells may be achieved by latent aggregation viaexpression of polypeptides from the target tissue cells, an examplebeing in the specific embodiment of the liver, facilitating activationof target immune receptors in the absence of a target disease antigen tofacilitate signaling.

C. Linker

In particular embodiments of the disclosure, there is a linker thatconnects at least one liver antigen-targeting entity with at least oneimmunostimulatory entity, for example on the same molecule. The linkeris configured to maintain proper geometry for efficacy and T cellactivation, in particular embodiments.

In some cases, the linker comprises sequence that is rich in glycineand/or serine. In specific cases, the linker comprises sequence that isat least 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% glycine and/or serine. Inspecific cases the linker also comprises one or more threonines. Thelinker may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more repeats of aseries of glycine and/or serine residues (for example, GGSG and/orGGGS). In specific embodiments, the linker is of a certain length, suchas at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, or150 amino acids in length. In other cases, the linker is no more than 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, or 150 amino acidsin length.

In some cases, one or more Fc domains are used in the linker, includingan Fc domain from IgG1, IgG2, IgG3, or IgG4, for example. Specificembodiments employ CH2 and CH3 domains from IgG, singly or incombination. In some cases, the Fc domain employed in the linker ismodified, and there may be one or more modifications. In particularembodiments, the modification(s) alters one or more of the followingproperties of the Fc domain: 1) to have reduced FcR (CD64, CD32, CD16,CD23, CD89) γ receptor binding; 2) to make them monomeric in structure;3) to remove complement activation 4) to remove FcRn binding; and/or 5)to have increased FcRy receptor binding.

In certain embodiments, one or more immunoglobulin domains are employedin the linker. The immunoglobulin domain(s) may come from any member ofthe Ig superfamily, in some embodiments. In specific embodiments, one ormore domains from CD86 and/or CD80, CD4 and/or CD8, are utilized. Thedomain may be an Ig variable-like (IgV) domain, an Ig constant-like(IgC) domains, and/or an intracellular domain, for example. In specificembodiments, when an Fc domain is utilized the CH2 and/or CH3 domain maybe replaced with an immunoglobulin domain, such as a domain from CD80,CD86, CD4 and/or CD 8, for example.

In specific aspects, the IgG Fc region comprises a) one or moremutations to disrupt FcR binding, b) a deletion of the CH2 domain,and/or c) replacement of the CH2 domain, for example with anotherimmunoglobulin domain from a different human protein (such as animmunoglobulin from an alternative human protein selected from human CD4domains D2 through D4). Mutations that disrupt FcR binding may belocated in the hinge region and/or glycosylation site of IgG Fc domain(including IgG1 Fc domain, IgG2 Fc domain, or IgG4 Fc domain, forexample.

Linker domain sequences that may be utilized in specific embodiments areas follows:

IgG1 (A A) Fc (linker domain or region, hinge-CH2-CH3, mutated FcRbinding) (SEQ ID NO: 31)EPKSCDKTHT CPPCPAPEAA GGPSVFLFPP KPKDTLMISR TPEVTCVVVDVSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLNGKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSLTCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKSRWQQGNVFSC SVMHEALHNH YTQKSLSLSP GKIgG2(AA) Fc (linker domain or region,hinge-CH2-CH3, mutated FcR binding) (SEQ ID NO: 32)ERKCCVECPP CPAPPAAAPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHEDPEVQFNWYV DGVEVHNAKT KPREEQFNST FRVVSVLTVV HQDWLNGKEY KCKVSKGLP APIEKTISKT KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDISVEWESNGQPEN NYKTTPPMLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMHEALHNHYTQK SLSLSPGK IgG4m Fc (linker domain or region, hinge-CH2-CH3,mutated FcR binding) (SEQ ID NO: 33)ESKYGPPCPS CPAPPVAGPS VFLFPPKPKD TLMISRTPEVTCVVVDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFQST YRVVSVLTVLHQDWLNGKEY KCKVSNKGLP SSIEKTISK KGQPREPQVY TLPPSQEEMTKNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSRLTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGKIgG1(AA) CH2 domain only (mutated cysteines inhinge domain to abrogate dimerization, andmutated hinge domain to abrogate FcR binding) (SEQ ID NO: 34)EPKSSDKTHTSPPSPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNK ALP APIEKTI SK AKGQPRE IgG2(AA) CH2 domain only (mutated cysteines in hinge domain to abrogate dimerization, andmutated hinge domain to abrogate FcR binding). (SEQ ID NO: 35)ERKCCVECPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPRE IgG1 wildtype sequence(SEQ ID NO: 36) EP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTPEVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDELTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSV MHEALHNHYT QK SLSLSPGK IgG4 wildtype sequence(SEQ ID NO: 37) ESKYGPPCPSCP APEFLGGPSV FLFPPKPKDT LMISRTPEVTCVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLHQDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTKNQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL T VDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK

D. Composition Molecules and Vectors

Embodiments of the disclosure encompass compositions that comprise atleast one molecule that comprises at least one liver antigen-targetingentity. Embodiments of the disclosure also encompass compositions thatcomprise at least one molecule that comprises at least oneimmunostimulatory entity. Embodiments of the disclosure encompasscompositions that comprise at least one molecule that comprises both ofat least one liver antigen-targeting entity and at least oneimmunostimulatory entity. In specific embodiments the molecule alsocomprises a linker that connects at least one liver antigen-targetingentity and at least one immunostimulatory entity. In cases wherein morethan one entity of any kind are comprised on the same molecule, they areoperably linked such that the more than one entity is capable ofperforming their respective functions, for example in conjunction withone another.

In some cases, the molecule comprising the one or more liverantigen-targeting entities and/or the one or more immunostimulatoryentities is a polynucleotide, including DNA or RNA. When two or moreentities are on a polynucleotide molecule, the regulation of expressionof the two or more entities may be coordinated, such as being the sameelement(s), for example, or they may utilize different regulatoryelements for regulation of expression. A “regulatory element” as usedherein refers to one or more transcriptional control elements, such asnon-coding cz's-acting transcriptional control elements, capable ofregulating and/or controlling transcription of an RNA from a codingregion, in particular tissue-specific transcription. Regulatory elementsmay comprise at least one transcription factor binding site, more inparticular at least one binding site for a tissue-specific transcriptionfactor, most particularly at least one binding site for a liver-specifictranscription factor. Regulatory elements may comprise enhancersequences. Regulatory elements may be situated either upstream (e.g. inthe promoter region) or downstream (e.g. in the 3′ UTR) of the sequencethey regulate in vivo, and may be located in the immediate vicinity ofthe gene or further away. In specific aspects, one or more regulatoryelements on the polynucleotide are tissue-specific, such asliver-specific regulatory sequences, for example. Examples of livertissue-specific regulatory elements include at least 1) thyroxinebinding globulin (TBG) promoter; and/or 2) a regulatory element asdescribed in US 2011/0184049, 3) albumin enhancer/promoter, 4) apoEpromoter, 5) alpha1-antitrypsin promoter, 6) HBV core promoter; and 7)combinations thereof.

In particular embodiments, a polynucleotide that expresses two or moreentities of any kind is configured such that the two or more entitiesare expressed as a fusion protein.

In specific examples, the polynucleotide encodes a sequence that allowsthe expressed polypeptide to be secretable from a cell, such as a leadersequence. The leader sequence is configured on the fusion proteinappropriately so that any cell in which the polynucleotide resides mayexpress the polypeptide and allow the polypeptide to become secreted sothat it may act upon other cells, for example. In specific embodiments,the leader sequence is about 5-30 amino acids long and is present at theN-terminus of the fusion protein. In at least some cases, a core of theleader sequence (which may also be referred to as a signal peptide)contains a long stretch of hydrophobic amino acids that has a tendencyto form a single alpha-helix. Examples in amino acid format include butare not limited to the following: MDWIWRILFLVGAATGAHS (SEQ ID NO: 22),MALPVTALLLPLALLLHAARP (SEQ ID NO:23), or MEFGLSWLFLVAILKGVQCSR (SEQ IDNO:24).

In some cases, a polynucleotide comprises at least one liverantigen-targeting entity and/or at least one immunostimulatory entityand in a 5′ to 3′ direction on the polynucleotide each of the entities(and more entities, where applicable) may be in any order so long asthey are capable of performing their respective function. In specificcases, in a 5′ to 3′ direction on the polynucleotide, the order on themolecule may be as follows, for example:

-   -   a) liver antigen-targeting entity-immunostimulatory entity;    -   b) liver antigen-targeting entity-linker-immunostimulatory        entity;    -   c) immunostimulatory entity-liver antigen-targeting entity; or    -   d) immunostimulatory entity-linker-liver antigen-targeting        entity.    -   e) immunostimulatory entity-linker    -   f) immunostimulatory entity

In specific cases, when the molecule encodes a bispecific antibody, theorder on the molecule in a 5′ to 3′ direction may be as follows, forexample:

-   -   a) scFv (liver antigen-targeting entity)-scFv (immunostimulatory        entity);    -   b) scFv (liver antigen-targeting entity)-linker-scFv        (immunostimulatory entity);    -   c) scFv (immunostimulatory entity)-scFv (liver antigen-targeting        entity); or    -   d) scFv (immunostimulatory entity)-linker-scFv (liver        antigen-targeting entity)

In specific cases, when the molecule comprises a cytokine, the order onthe molecule in a 5′ to 3′ direction may be as follows, for example:

-   -   a) scFv (liver antigen-targeting entity)-cytokine;    -   b) scFv (liver antigen-targeting entity)-linker-cytokine;    -   c) cytokine-scFv (liver antigen-targeting entity); or    -   d) cytokine-linker-scFv (liver antigen-targeting entity)

In specific embodiments, the molecule comprising the one or more liverantigen-targeting entities and/or the one or more immunostimulatoryentities is a polypeptide. In particular embodiments, the polypeptide issecretable.

In particular embodiments, the polynucleotides and/or polypeptidesencompassed by the disclosure are provided to an individual in nakedform. However, in other embodiments the polynucleotides and/orpolypeptides encompassed by the disclosure are configured in and/or on avector. The vector may of any kind and in at least some cases acts toprotect the polynucleotide and/or polypeptide of which it contains, oris attached to, from one or more deleterious events and/or environments(such as nucleases or proteases, respectively), for example. Any viralor non-viral vector may be used in vivo or ex vivo to deliver thepolynucleotides into target cells, including liver cells, such as liverdiseased cells that includes pathogen-infected cells and tumor cellsand/or cells within the tumor microenvironment. This includes, but isnot limited to, adenovirus (replication competent, replicationincompetent, helper dependent), adeno associated virus (AAV) (see, forexample, US 2002/0151509, which is incorporated by reference herein inits entirety), Herpes simplex virus 1 (HSV1), myxoma virus, reovirus,poliovirus, vesicular stomatitis virus (VSV), measles virus (MV),Newcastle disease virus (NDV), retroviruses, nanoparticles, cationiclipids, cationic polymers, lipid nanoparticles, liposomes and/or lipidpolymers, for example. The polynucleotide may be generated as part ofthe same molecule as a vector, the polynucleotide may be encompassedwithin a vector, and/or the polynucleotide may be attached to a vector,as examples.

Thus, the vector may be viral or non-viral, in certain cases. Anon-viral vector may comprise a plasmid, liposomes, nanoparticles,microbubble plus ultrasound, dendrimers, cationic magneticnanoparticles, lipoplexes (lipid-based); inorganic molecules, etc. Aviral vector may be of any kind, but in specific embodiments the viralvector is an adenoviral vector, an adeno-associated viral vector, aretroviral vector, or a lentiviral vector. In specific cases, a viralvector may comprise one or more modifications that change a property ofthe viral vector. In cases wherein the viral vector is anadeno-associated viral vector, the adeno-associated virus may compriseone or more modifications that change a property of the adeno-associatedvirus, such as a capsid mutation, for example, that renders it topreferentially transduce a target tissue or organ, such as a liver. Anadeno-associated viral vector may be utilized in some cases because itpreferentially transduces a certain target tissue or organ, such as aliver. In specific embodiments, the adeno-associated viral vector is ofthe serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,AAV10, AAV11, AAV 12, or an AAV serotype isolated from a non-humanprimate.

In specific cases, when the polynucleotide is RNA, such as mRNA, thevector may comprise a lipid-based nanoparticle. In certain cases whereinthe polynucleotide is an mRNA, it may or may not comprise one or moremodified nucleotides (i.e., with additional chemical groups behindcanonical ribonucleotides, with pseudouridine and 5-methylcytosine beingexamples) that increase translation and/or inhibit the innate immuneresponse in an individual being provided the mRNA. In preferredembodiments, the mRNA molecule contains one or more of, or all of, a 5′guanosine cap, a 5′ UTR, the open reading frame, a 3′ UTR, and a polyAtail. The sequence of the 3′ UTR may be manipulated to add sequencestargeted by host miRNA's, thereby offered a level of expression controlto either diseased cells or to normal cells.

In specific embodiments, a vector comprises at least one expressionconstruct that encodes a molecule that comprises at least one liverantigen-targeting entity and at least one immunostimulatory entity. Anexpression construct may comprise coding regions that encode the atleast one liver antigen-targeting entity and the at least oneimmunostimulatory entity, and in some cases the entities are regulatedin the expression construct by one or more regulatory regions. Althoughin certain cases they share at least one regulatory region, such as bybeing expressed as a fusion protein, in alternative cases they utilizedifferent regulatory regions. In any case, a regulatory region in theexpression cassette may be tissue-specific, including liver-specific.

The non-natural polynucleotide and/or polypeptide compositionsencompassed by the disclosure may be manufactured by any suitable means.In specific embodiments, they are generated using standard recombinationmeans in the art. Basic procedures for constructing recombinant DNA andRNA molecules in accordance with the present disclosure are disclosed innumerous publications, including Sambrook et al, In: Molecular Cloning:A Laboratory Manual, Second Edition, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (1989), which is herein incorporated by reference.In particular cases, the compositions are stored under suitableconditions and provided to an individual (including through a medicalpractitioner) at a time of need. E. Specific Examples of Compositions

Particular examples of compositions having specific scFvs and linkersare as follows:

19-Fc-CD3: Leader-scFv XTL19-IgGI Fc domain-OKT3 scFv binding human CD3(SEQ ID NO: 15) MDWIWRILFL VGAATGAHSQ VQLVESGGGV VQPGGSLRLS CAPSGFVFRSYGMHWVRQTP GKGLEWVSLI WHDGSNRFYA DSVKGRFTIS RDNSKNTLYLQMNSLRAEDT AMYFCARERL IAAPAAFDLW GQGTLVTVSS GGGGSGGGGSGGGGSSYVLT QPPSVSVAPG KTARISCGGN NIGTKNVHWY QQKPGQAPVLVVYADSDRPS GIPERFSGSN SGNTATLTIS RVEVGDEADY YCQVWDSVSYHVVFGGGTTL TVLGSGGGGS DKTHTCPPCP APELLGGPSV FLFPPKPKDTLMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTYRVVSVLTVLH QDWLNGKEYK CKVS KALPA PIEKTISKAK GQPREPQVYTLPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDSDGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGKSSDIKLQQSGAEL ARPGASVKMS CKTSGYTFTR YTMHWVKQRP GQGLEWIGYI PSRGYTNYN QKFKDKATLT TDKSSSTAYM QLSSLTSEDS AVYYCARYYDDHYCLDYWGQ GTTLTVSSGG GGSGGGGSGG GGSDIQLTQS PAFMSASPGEKVTMT CRASS SVSYMNWYQQ KSGTSPKRWI YDTSKVASGV PYRFSGSGSGTSYSLTISSM EAEDAATYYC19-mFc-CD3: Leader-scFv XTL19-human IgGI Fc domain withmutations for monomeric form and for abrogated Fcreceptor binding-OKT3 scFv binding human CD3 (SEQ ID NO: 16)MDWIWRILFL VGAATGAHSQ VQLVESGGGV VQPGGSLRLS CAPSGFVFRSYGMHWVRQTP GKGLEWVSLI WHDGSNRFYA DSVKGRFTIS RDNSKNTLYLQMNSLRAEDT AMYFCARERL IAAPAAFDLW GQGTLVTVSS GGGGSGGGGSGGGGSSYVLT QPPSVSVAPG KTARISCGGN NIGTKNVHWY QQKPGQAPVLVVYADSDRPS GIPERFSGSN SGNTATLTIS RVEVGDEADY YCQVWDSVSYHVVFGGGTTL TVLGSGGGGS GAPPVAGPSV FLFPPKPKDT LMISRTPEVTCVVVGVSHED PEVKFNWYVD GVEVHNAKTK PREEQYQSTY RVVSVLTVLHQDWLNGKEYK CAVSNKQLPS SIEKTISKAK GQPREPQVYT KPPSRDELTKNQVSLSCLVK GFYPSDIAVE WESNGQPENN YKTTVPVLDS DGSFRLASYLTVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGKGSG GGGSDIKLQQ S GAELARPGA SVKMSCKTSG YTFTRYTMHW VKQRPGQGLE WIGYINPSRGYTNYNQKFKD KATLTTDKSS STAYMQLSSL TSEDSAVYYC ARYYDDHYCLDYWGQGTTLT VSSGGGGSGG GGSGGGGSDI QLTQSPAFMS ASPGEKVTMTPKRWIYDTSK CRASSSVSYM NWYQQKSGTS VASGVPYRFS GSGSGTSYSLTISSMEAEDA ATYYCQQWSS19-G4m-CD3: Leader-scFv XTL19-human IgG4 Fc domain withabrogated Fc receptor binding-OKT3 scFv binding human CD3(SEQ ID NO: 17) MDWIWRILFL VGAATGAHSQ VQLVESGGGV VQPGGSLRLS CAPSGFVFRSYGMHWVRQ TPGKGLEWVS LIWHDGSNRF YADSVKGRFT ISRDNSKNTLYLQMNSLR AEDTAMYF CARERLIAAP AAFDLWGQGT LVTVSSGGGGSGGGGSGGGG SSYVLTQPPS VSVAPGKTAR ISCGGNNIGT KNVHWYQQKPGQAPVLVVYA DSDRPSGIPE RFSGSNSGNT ATLTISRVEV GDEADYYCQVWDSVSYHVVF GGGTTLTVLG SGGGGSESKY GPPCPSCPAP PVAGPSVFLFPPKPKDTLM ISRTPEVTC VVVDVSQEDP EVQFNWYVDG VEVHNAKTKPREEQFQSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKGLPSS IEKTISKAKGQPREPQVYTL PPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPENNYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVM HEALHNHYTQKSLSLSPGK GSGGGGSDIK LQQSGAELAR PGASVKMSCK TSGYTFTRYTMHWVKQRPGQ GLEWIGYINP SRGYTNYNQK FKDKATLTTD KSSSTAYMQL19-CD3: Leader-scFv XTL19-short glycine serine linker-OKT3 scFvbinding human CD3 (SEQ ID NO: 18)MDWIWRILFL VGAATGAHSQ VOLVESGGGV VQPGGSLRLS CAPSGFVFRSYGMHWVRQTP GKGLEWVSLI WHDGSNRFYA DSVKGRFTIS RDNSKNTLYLQMNSLRAEDT AMYFCARERL IAAPAAFDLW GQGTLVTVSS GGGGSGGGGSGGGGSSYVLT QPPSVSVAPG KTARISCGGN NIGTKNVHWY QQKPGQAPVLVVYADSDRPS GIPERFSGSN SGNTATLTIS RVEVGDEADY YCQVWDSVSYHVVFGGGTTL TVLGSGGGGS DIKLQQSGAE LARPGASVKM SCKTSGYTFTRYTMHWVKQR PGQGLEWIGY INPSRGYTNY NQKFKDKATL TTDKSSSTAYMQLSSLTSED SAVYYCARYY DDHYCLDYWG QGTTLTVSSG GGGSGGGGSGGGGSDIQLTQ SPAIMSASPG EKVTMTCRAS SSVSYMNWYQ QKSGTSPKRWIYDTSKVASG VPYRFSGSGS GTSYSLTISS MEAEDAATYY CQQWSSNPLT FGAGTKLELK SCD3-Fc-19: Leader-OKT3 scFv binding human CD3-IgG1 Fc domain-scFv XTL19(SEQ ID NO: 19) MDWIWRILFL VGAATGAHSD IKLQQSGAEL ARPGASVKMS CKTSGYTFTRYTMHWVKQRP GOGLEWIGYI NPSRGYTNYN QKFKDKATLT TDKSSSTAYMQLSSLTSEDS AVYYCARYYD DHYCLDYWGQ GTTLTVSSGG GGSGGGGSGGGGSDIQLTQS PAIMSASPGE KVTMTCRASS SVSYMNWYQQ KSGTSPKRWIYDTSKVASGV PYRFSGSGSG TSYSLTISSM EAEDAATYYC QQWSSNPLTFGAGTKLELKS SGGGGSDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMISRTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVSVLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPSRDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSFFLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGKSSDIQVQLVESGGGVVQ PGGSLRLSCA PSGFVFRSYG MHWVRQTPGK GLEWVSLIWHDGSNRFYADS VKGRFTISRD NSKNTLYLQM NSLRAEDTAM YFCARERLIAAPAAFDLWGQ GTLVTVSSGG GGSGGGGSGG GGSSYVLTQP PSVSVAPGKTARISCGGNNI GTKNVHWYQQ KPGQAPVLVV YADSDRPSGI PERFSGSNSGNTATLTISRV EVGDEADYYCCD3-B7.1-19: Leader-OKT3 scFv binding human CD3-human CD80ectodomain-scFv XTL19 (SEQ ID NO: 20)MDWIWRILFL VGAATGAHSD IKLQQSGAEL ARPGASVKMS CKTSGYTFTRYTMHWVKQRP GOGLEWIGYI NPSRGYTNYN QKFKDKATLT TDKSSSTAYMQLSSLTSEDS AVYYCARYYD DHYCLDYWGQ GTTLTVSSGG GGSGGGGSGGGGSDIQLTQS PAIMSASPGE KVTMTCRASS SVSYMNWYQQ KSGTSPKRWIYDTSKVASGV PYRFSGSGSG TSYSLTISSM EAEDAATYYC QQWSSNPLTFGAGTKLELKS SGGGGSPYLN FFQLLVLAGL SHFCSG VIHV TKEVKEVATLSCGHNVSVEE LAQTRIYWQK EKKMVLTMMS GDMNIWPEYK NRTIFDITNNLSIVILALRP SDEGTYECVV LKYEKDAFKR EHLAEVTLSV KADFPTPSIS DFEIPTSNIRRIICSTSGGF PEPHLSWLEN GEELNAINTT VSQDPETELY AVS SKLDFNMTTNHSFMCLI KYGHLRVNQT FNSSDIQVQL VESGGGVVQP GGSLRLSCAPSGFVFRSYGM HWVRQTPGKG LEWVSLIWHD GSNRFYADSV KGRFTISRDNSKNTLYLQMN SLRAEDTAMY FCARERLIAA PAAFDLWGQG TLVTVSSGGGGSGGGGSGGG GSSYVLTQPP SVSVAPGKTA RISCGGNNIG TKNVHWYQQKPGQAPVLVVY ADSDRPSGIP ERFSGSNSGN TATLTISRVE VGDEADYYCQ VWDSVSYHVVCD 3-B7.1-G4m-19: Leader-OKT3 scFv binding human CD3-humanCD80 ectodomain-human IgG4 Fc domain with abrogatedFc receptor binding-scFv XTL19 (SEQ ID NO: 21)MDWIWRILFL VGAATGAHSD IKLQQSGAEL ARPGASVKMS CKTSGYTFTRYTMHWVKQRP GQGLEWIGYI NPSRGYTNYN QKFKDKATLT TDKSSSTAYMQLSSLTSEDS AVYYCARYYD DHYCLDYWGQ GTTLTVSSGG GGSGGGGSGGGGSDIQLTQS PAIMSASPGE KVTMTCRASS SVSYMNWYQQ KSGTSPKRWIYDTSKVASGV PYRFSGSGSG TSYSLTISSM EAEDAATYYC QQWSSNPLTFGAGTKLELKS SGGGGSPYLN FFQLLVLAGL SHFCSGVIHV TKEVKEVATLSCGHNVSVEE LAQTRIYWQK EKKMVLTMMS GDMNIWPEYK NRTIFDITNNLSIVILALRP SDEGTYECVV LKYEKDAFKR EHLAEVTLSV KADFPTPSIS DFEIPTSNIRRIICSTSGGF PEPHLSWLEN GEELNAINTT VSQDPETELY AVS SKLDFNMTTNHSFMCLI KYGHLRVNQT FNSGGGSESK YGPPCPSCPA PPVAGPSVFLFPPKPKDTLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPREEQFQSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQPREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIA VEWE SNGQPENNYKTTPPVLDSDG SFFLYSRLTV DK SRWQEGN V FSCSVMHEAL HNHYTQKSLSLSPGKSSDIQ VQLVESGGGV VQPGGSLRLS CAPSGFVFRS YGMHWVRQTPGKGLEWVSLI WHDGSNRFYA DSVKGRFTIS RDNSKNTLYL QMNSLRAEDTAMYFCARERL IAAPAAFDLW GQGTLVTVSS GGGGSGGGGS GGGGSSYVLTQPPSVSVAPG KTARISCGGN NIGTKNVHWY QQKPGQAPVL VVYADSDRPSGIPERFSGSN SGNTATLTIS RVEVGDEADY YCQVWDSVSY HVVFGGGTTLLeader-19-mFc-hCD3 = 19.45.9 or XTL-19 scFv-IgG1m CH2-CH3domain with Fc mutation-humanized OKT3 (gOKT3-7) (SEQ ID NO: 38)MDWIWRILFL VGAATGAHSQ VOLVESGGGV VQPGGSLRLS CAPSGFVFRSYGMHWVRQTP GKGLEWVSLI WHDGSNRFYA DSVKGRFTIS RDNSKNTLYLQMNSLRAEDT AMYFCARERL IAAPAAFDLW GQGTLVTVSS GGGGSGGGGSGGGGSSYVLT QPPSVSVAPG KTARISCGGN NIGTKNVHWY QQKPGQAPVLVVYADSDRPS GIPERFSGSN SGNTATLTIS RVEVGDEADY YCQVWDSVSYHVVFGGGTTL TVLGEPKSCD KTHTCPPCPA PEAAGGPSVF LFPPKPKDTLMISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYQSTYRVVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTLPPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSDGSFFLYSKLT VDKSRWQQGN VESCSVMHEA LHNHYTQKSL SLSPGKSGSGSQVQLVQSGG GVVQPGRSLR LSCKASGYTF TRYTMHWVRQ APGKGLEWIGYINPSRGYTN YNQKVKDRFT ISRDNSKNTA FLQMDSLRPE DTGVYFCARYYDDHYCLDYW GQGTPVTVSS GGGGSGGGGS GGGGSDIQMT QSPSSLSASVGDRVTITCSA SSSVSYMNWY QQTPGKAPKR WIYDTSKLAS GVPSRFSGSGSGTDYTFTIS SLQPEDIATY YCQQWSSNPF TFGQGTKLQI TRLeader-19-mFc = 19.45.9 or XTL-19 scFv-IgG1m CH2-CH3 domainwith Fc mutation (SEQ ID NO: 39)MDWIWRILFL VGAATGAHSQ VOLVESGGGV VQPGGSLRLSCAPSGFVFRS YGMHWVRQTP GKGLEWVSLI WHDGSNRFYA DSVKGRFTISRDNSKNTLYL QMNSLRAEDT AMYFCARERL IAAPAAFDLW GQGTLVTVSSGGGGSGGGGS GGGGSSYVLT QPPSVSVAPG KTARISCGGN NIGTKNVHWYQQKPGQAPVL VVYADSDRPS GIPERFSGSN SGNTATLTIS RVEVGDEADYYCQVWDSVSY HVVFGGGTTL TVLGEPKSCD KTHTCPPCPA PEAAGGPSVFLFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKPREEQYQSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKGQPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNYKTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGKLeader-19-hCD3 = 19.79.5 or XTL-19 scFv-humanized OKT3 (gOKT3-7)(SEQ ID NO: 40)  MDWIWRILFL VGAATGAHSQ VOLVESGGGV VQPGGSLRLSCAPSGFVFRS YGMHWVRQTP GKGLEWVSLI WHDGSNRFYA DSVKGRFTISRDNSKNTLYL QMNSLRAEDT AMYFCARERL IAAPAAFDLW GQGTLVTVSSGGGGSGGGGS GGGGSSYVLT QPPSVSVAPG KTARISCGGN NIGTKNVHWYQQKPGQAPVL VVYADSDRPS GIPERFSGSN SGNTATLTIS RVEVGDEADYYCQVWDSVSY HVVFGGGTTL TVLGSGGGGS VQLVQSGGGV VQPGRSLRLSCKASGYTFTR YTMHWVRQAP GKGLEWIGYI NPSRGYTNYN QKVKDRFTISRDNSKNTAFL QMDSLRPEDT GVYFCARYYD DHYCLDYWGQ GTPVTVSSGGGGSGGGGSGG GGSDIQMTQS PSSLSASVGD RVTITCSASS SVSYMNWYQQTPGKAPKRWI YDTSKLASGV PSRFSGSGSG TDYTFTISSL QPEDIATYYCQQWSSNPFTF GQGTKLQITRLeader-17-mFc-hCD3 = 17.1.41 or XTL-17 scFv-IgG1m CH2-CH3domain with Fc mutation-humanized OKT3 (gOKT3-7) (SEQ ID NO: 41)MDWIWRILFL VGAATGAHSQ VOLVESGGGV VRPGRSLRLSCAASGFAFSD YSINWVRQAP GKGLEWVAII SYDGRITYYR DSVKGRFTISRDDSKNTLYL QMNSLRTEDT AVYYCARQYY DFWSGSSVGR NYDGMDVWGLGTTVTVSSGG GGSGGGGSGG GGSDIVMTQS PLSLSVTPGE PASISCRSSQSLLHRSGNNY LDWYLQKPGH SPQLLIYVGS NRASGVPDRF SGSGSGTEYTLRISTVEAED VGVYYCMQAL QTPRTFGQGT KLEIKREPKS CDKTHTCPPCPAPEAAGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYVDGVEVHNAKT KPREEQYQST YRVVSVLTVL HQDWLNGKEY KCKVSNKALPAPIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAVEWESNGOPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMHEALHNHYTQK SLSLSPGKSG SGSQVQLVQS GGGVVQPGRS LRLSCKASGYTFTRYTMHWV RQAPGKGLEW IGYINPSRGY TNYNQKVKDR FTISRDNSKNTAFLQMDSLR PEDTGVYFCA RYYDDHYCLD YWGQGTPVTV SSGGGGSGGGGSGGGGSDIQ MTQSPSSLSA SVGDRVTITC SASSSVSYMN WYQQTPGKAPKRWIYDTSKL ASGVPSRFSG SGSGTDYTFT ISSLQPEDIA TYYCQQWSSN PFTFGQGTKL QITRLeader-17-mFc = 17.1.41 or XTL-17 scFv-IgG1m I CH2-CH3 domainwith Fc mutation (SEQ ID NO: 42)MDWIWRILFL VGAATGAHSQ VQLVESGGGV VRPGRSLRLSCAASGFAFSD YSINWVRQAP GKGLEWVAII £ SYDGRITYYR DSVKGRFTISRDDSKNTLYL QMNSLRTEDT AVYYCARQYY DFWSGSSVGR NYDGMDVWGLGTTVTVSSGG GGSGGGGSGG GGSDIVMTQS PLSLSVTPGE PASISCRSSQSLLHRSGNNY LDWYLQKPGH SPQLLIYVGS NRASGVPDRF SGSGSGTEYTLRISTVEAED VGVYYCMQAL QTPRTFGQGT KLEIKREPKS CDKTHTCPPCPAPEAAGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYVDGVEVHNAKT KPREEQYQST YRVVSVLTVL HQDWLNGKEY KCKVSNKALPAPIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAVEWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMHEALHNHYTQK SLSLSPGK Leader-17-hCD3 = 17.1.41 or XTL-17 scFv-humanizedOKT3 (gOKT3-7) (SEQ ID NO: 43)MDWIWRILFL VGAATGAHSQ VOLVESGGGV VRPGRSLRLSCAASGFAFSD YSINWVRQAP GKGLEWVAII SYDGRITYYR DSVKGRFTISRDDSKNTLYL QMNSLRTEDT AVYYCARQYY DFWSGSSVGR NYDGMDVWGLGTTVTVSSGG GGSGGGGSGG GGSDIVMTQS PLSLSVTPGE PASISCRSSQSLLHRSGNNY LDWYLQKPGH SPQLLIYVGS RASGVPDRF SGSGSGTEYTLRISTVEAED VGVYYCMQAL QTPRTFGQGT KLEIKRSGGG GSQVQLVQSGGGVVQPGRSL RLSCKASGYT FTRYTMHWVR QAPGKGLEWI GYINPSRGYTNYNQKVKDRF TISRDNSKNT AFLQMDSLRP EDTGVYFCAR YYDDHYCLDYWGQGTPVTVS SGGGGSGGGG SGGGGSDIQM TQSPSSLSAS VGDRVTITCSASSSVSYMNW YQQTPGKAPK RWIYDTSKLA SGVPSRFSGS GSGTDYTFTISSLQPEDIAT YYCQQWSSNP FTFGQGTKLQ ITRLeader-hCD3-mFc = leader-humanized OKT3 (gOKT3-7)-mFc(mutations in Fc domain against Fc receptor proteins) (SEQ ID NO: 44)MDWIWRILFL VGAATGAHSQ VQLVQSGGGV VQPGRSLRLSCKASGYTFTR YTMHWVRQAP GKGLEWIGYI NPSRGYTNYN QKVKDRFTISRDNSKNTAFL QMDSLRPEDT GVYFCARYYD DHYCLDYWGQ GTPVTVSSGGGGSGGGGSGG GGSDIQMTQS PSSLSASVGD RVTITCSASS SVSYMNWYQQTPGKAPKRWI YDTSKLASGV PSRFSGSGSG TDYTFTISSL QPEDIATYYCQQWSSNPFTF GQGTKLQITR EPKSCDKTHT CPPCPAPEAA GGPSVFLFPPKPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQYQSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPREPQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIA VEWESNG QPENNYKTTPPVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GKLeader-hCD3 = leader-humanized OKT3 (gOKT3-7) (SEQ ID NO: 45)MDWIWRILFL VGAATGAHSQ VQLVQSGGGV VQPGRSLRLSCKASGYTFTR YTMHWVRQAP GKGLEWIGYI NPSRGYTNYN QKVKDRFTISRDNSKNTAFL QMDSLRPEDT GVYFCARYYD DHYCLDYWGQ GTPVTVSSGGGGSGGGGSGG GGSDIQMTQS PSSLSASVGD RVTITCSASS SVSYMNWYQQTPGKAPKRWI YDTSKLASGV PSRFSGSGSG TDYTFTISSL QPEDIATYYCQQWSSNPFTF GQGTKLQITR

In particular embodiments, provided herein are specific examples ofpolynucleotide compositions that encode at least one liverantigen-targeting entity and/or immunostimulatory entity, and/or thepolypeptides themselves. In certain compositions, the liverantigen-targeting entity consists of or comprises a single chainantibody, a single chain variable fragment, a camelid antibody, or apeptide. An immunostimulatory domain may comprise an anti-CD3 scFv, ananti-CD28 scFv, anti-41BB scFv, anti-OX40 scFv, anti-CTLA4 scFv, ananti-CD 16 scFv, anti-PD1 scFv, anti-PD-L1 scFv, anti-CD47 scFv, part orall of the ectodomain for a ligand for CD28 (such as part or all of theectodomain of CD80 and/or CD86), part or all of the ectodomain of 4 IBBligand, part or all of the ectodomain of the LIGHT protein, ICOS-ligand,CD276 (B7-H3), B7-H4, and B7-H6, CD134L, CD137L, or a cytokine (IL-2,IL-15, etc.).

Any of such specific compositions, or others, may reside in a vector oras part of a vector or attached thereto, for example, and in specificcases the vector is an adeno-associated virus, wherein the virus has acassette that encodes a tissue specific promoter, in order to targetexpression to a specific organ. When the vector comprises messenger RNA,the mRNA may be delivered in a lipid-based nanoparticle, and any mRNAencompassed by the disclosure may have modified nucleotides to increasetranslation and inhibit the innate immune response.

In certain vector embodiments, there is an adeno-associated virus with amutated capsid or serotype that preferably transduced human liver. Anexample is an AAV with serotype preferably AAV8 or AAV9. Some vectorsmay utilize the TBG promoter, for example for targeting expression ofbispecific antibodies to the liver after delivery by AAV.

Bispecific antibodies that target a cell surface antigen on a diseasedcell are encompassed herein, including those that at least target HBVsmall surface antigen, HBV middle surface antigen, HBV large surfaceantigen, HCV E1 protein, HCV E2 protein, EBV glycoprotein, CMVglycoprotein, EphA2, glypican-3, HER2, PSCA, TEM8, CD 19, EGFRvIII, etc.In specific embodiments, there is a bispecific antibody construct thatcomprises on a molecule the orientation: scFv (target)-linker-scFv(immune) or scFv (immune)-linker-scFv (target). In other embodiments,there is an immunocytokine construct comprising the orientation:cytokine-linker-scFv (target) or scFv (target)-linker-cytokine.

In alternative embodiments, the composition provided to an individualcomprises an immunostimulatory protein alone (i.e., anti-CD3-Fc,anti-CD3 scFv alone, anti-CD28-Fc, anti-CD28 scFv alone, or B7-Fc alone,or anti-PD1 alone) in the absence of a liver antigen-targeting entity,particularly absent on the same molecule, for example. Normally, thesemolecules would not be activating, but the in situ tissue expressiondescribed in this invention results in unexpected aggregation andactivation properties. In other alternative embodiments, the compositioncomprises a protein that comprises an antigen-targeting domain alone (ora polynucleotide encoding same), and in specific embodiments thecomposition with the antigen-targeting domain may be used in combinationwith an immunostimulatory ligand.

In alternative embodiments, the composition provided to an individualcomprises an additional cell protective polynucleotide sequence inaddition to the disease antigen targeting and/or immune stimulatorycomponents. The cell protective polynucleotide sequence will serve toinhibit the targeted tissue from suffering cytotoxic death amidst theinduced inflammation, preventing loss of normal cells and tissue whilepreserving therapeutic efficacy against the pathogen or cancer. Examplesof a cytoprotective agent may include an mRNA encoding the Bcl2, Bcl-XL,Mc1-1, CED-0, Bfl-1, X-linked inhibitor of apoptosis protein (XIAP),c-IAP1, C-IAP2, NAIP, Livin, Survivin, serpin proteinase inhibitor 9, orSERPINB4. Other examples include an siRNA, antisense oligonucleotide, ora morpholino targeting the knockdown of Fas receptor, T Falpha receptor,Bax, Bid, Bak, or Bad, genes that otherwise induce apoptosis. Acytoprotective agent may be an apoptosis inhibitor, in specific cases.In some cases the secretable polypeptide and the cytoprotective agentare encoded on the same nucleic acid molecule, for example if they wereregulated by separate promoters, or separated by an IRES or 2A element.In cases wherein the secretable polypeptide and cytoprotective agent areon separate nucleic acids, they can be delivered separately on twoseparate molecules, but packaged together in the same composition, suchas a nanoparticle.

II. Method of Use

Embodiments of the disclosure include methods of treating at least onemedical condition that affects a targeted tissue, such as the liver, ofa mammal, including humans, dogs, cats, horses, cows, pigs, sheep, etc.In specific embodiments, the disease is caused by a pathogen, althoughalternatively or additionally at least in part it may be environmentaland/or genetic in nature. In specific embodiments, the method isemployed for treating cancer or an infectious disease in a targetedtissue, including the liver. In specific embodiments, methods of thedisclosure comprise administering RNA or DNA that encodes proteins viaviral or non-viral vectors, although in alternative embodimentspolypeptides are administered.

Any disease in a specific tissue that can be treated with targetedtherapy may be treated with methods of the disclosure. However, inspecific embodiments a liver disease is treated, including any liverdisease. Viral liver diseases may be treated using targeted therapies ofthe disclosure, as well as cancers of the liver may be treated,including both primary and metastatic lesions. Specific liver diseasesinclude but are not limited to Hepatitis B infection, Hepatitis Ainfection, Hepatitis C infection, Hepatoblastoma, HepatocellularCarcinoma, and metastatic cancer of the breast, prostaste, pancreas,colon, rectum, esophagus, stomach, lungs, kidney, or skin, as examples.

Polynucleotides of the disclosure encode fusion proteins that bind atargeted antigen and that stimulate an immune function, and the fusionprotein(s) may be generated for and/or in an individual in any manner.In some cases, the polynucleotide is delivered to the individual locallysuch that upon delivery of the polynucleotide composition to thetargeted tissue or organ in vivo, the polynucleotide is taken up by thetissue or organ, and the fusion protein is produced in those cells.Following production of the fusion protein in the cells, the cellssecrete the fusion protein such that it is soluble and can bind itstarget(s) on other cells, including at least non-transduced cells, suchas diseased cells including pathogen-infected or cancer cells.

Delivery of a composition encompassed by the disclosure may be of anykind, route, duration, recurrence, and so forth. In particularembodiments delivery of the compositions is local in nature, although inalternative embodiments the delivery is systemic. In some cases the samecomposition is delivered to an individual in need thereof, although inother cases different compositions are provided to an individual in needthereof, whether it occurs at the same or different times. In specificembodiments, delivery of one or more compositions to an individual inneed thereof is in the absence of systemic delivery, such as in theabsence of constant infusion, for example.

In some methods of the disclosure, more than one composition comprisingat least one liver antigen-targeting entity and/or at least oneimmunostimulatory entity are provided to an individual, and in somecases the compositions are non-identical. They may be targetingdifferent liver antigens, they may be providing immunostimulationthrough different targets, or both, for example. In such cases, the twoor more different compositions may be provided to the individual at thesame time and/or at different times. In some aspects, differentcompositions are provided at different times over the course of suitabledurations in the span of time of delivery, including on the order of1-60 minutes, 1-24 hours, 1-7 days, 1-4 weeks, 1-12 months, or 1 or moreyears. In specific cases, a non-identical composition is provided to theindividual under certain treatment outcomes, such as when a particulartherapy becomes refractory (for example, with primary liver ormetastatic cancer). The administration of the composition(s) of thedisclosure is useful for all stages and types of cancer that affects theliver (as an example), including for minimal residual disease, earlycancer, advanced cancer, metastatic cancer and/or refractory cancer, forexample.

Particular embodiments of methods of use include delivery of bispecificantibodies to the liver using viral DNA or non-viral RNA vectors, forexample, as a platform for their expression. In specific embodiments,one can employ an off-the-shelf immunotherapy, including that having ahigher safety and efficacy index than infusion of recombinant bispecificantibody therapy, for example.

By way of illustration, diseased individuals or individuals suspected ofhaving disease or at risk therefore may be treated as described herein.The polynucleotides and/or polypeptides as described herein may beadministered to the individual and retained for extended periods oftime. The individual may receive one or more administrations of thepolynucleotides. In some embodiments, polynucleotides are encapsulatedto inhibit immune recognition and placed at the site of the tissue ororgan (or tumor, for cancer embodiments).

In various embodiments the expression constructs, nucleic acidsequences, vectors, host cells and/or pharmaceutical compositionscomprising the same are used for the prevention, treatment oramelioration of a liver disease. In particular embodiments, thepharmaceutical composition of the present disclosure may be particularlyuseful in preventing, ameliorating and/or treating disease related tothe liver, including viral infection or liver cancer having solidtumors, for example.

As used herein “treatment” or “treating,” includes any beneficial ordesirable effect on the symptoms or pathology of a disease orpathological condition, and may include even minimal reductions in oneor more measurable markers of the disease or condition being treated,e.g., pathogen infection or cancer. Treatment can involve either thereduction or amelioration of symptoms of the disease or condition and/orthe delaying of the progression of the disease or condition. “Treatment”does not necessarily indicate complete eradication or cure of thedisease or condition, or associated symptoms thereof.

In particular embodiments, the present disclosure contemplates, in part,polypeptides, nucleic acid molecules and/or vectors that can beadministered either alone or in any combination with another therapy,and in at least some aspects, together with a pharmaceuticallyacceptable carrier or excipient. In certain embodiments, nucleic acidmolecules or vectors may be stably integrated into the genome of thetargeted cells. In specific embodiments, viral vectors may be used thatare specific for certain cells or tissues and persist in said cells.Suitable pharmaceutical carriers and excipients are well known in theart. The compositions prepared according to the disclosure can be usedfor the prevention or treatment or delaying the above identifieddiseases.

Furthermore, the disclosure relates to a method for the treatment oramelioration of a liver disease comprising the step of administering toa subject in need thereof an effective amount of polynucleotides, cells,and/or vector(s), as contemplated herein and/or produced by a process ascontemplated herein.

In one embodiment, there is a method of making functional bi-, tri- orquadra-specific antibodies comprising administering two or more codingsequences targeting different antigens or immunostimulatory domains intoan individual, wherein the proteins generated may dimerize at randomgenerating a substantial fraction of antibodies with heterotypicpartners, thereby generating antibodies with dual target specificityand/or dual immune stimulatory properties. More specifically, antibodieswith Fc domains will naturally form dimers at the Fc-Fc interface.Expressing two or more antibodies in the same cell will result in randompairing of Fc domains, resulting in multiple antigen targeting withinthe same protein. Such proteins may be of use for higher affinitytargeting of cancer or pathogen cells via targeted two antigens at onetime. Similarly, one molecule targeting two immune molecules can lead tohigher activation and potency. The expression of two coding sequencesdelivered into a specific tissue such as the liver, where one antibodytargets a disease antigen and one antibody targets an immunostimatorymolecule, can also more simply make bispecific antibodies in situ in theliver in addition to the monospecific antibodies from anotherembodiment. This is an advantageous way to make multiple different typesof antibodies within a specific tissue, including bispecific antibodiesthat can redirect immune cells. In some embodiments, the Fc domains mayhave mutations to bias the pairing of two different proteins, in orderto have more heterologous Fc pairing.

In one embodiment, these multiple epitope targeting antibodies can begenerated in vivo in the liver tissue. Multiple different sequences areinfused at substantially the same time targeting multiple differentepitopes into the liver, leading a fraction of their Fc domains to bindto each other. As applied to the treatment of Hepatitis B virus, forexample, scFv's targeting a conformation and/or linear epitopes on smallsurface antigen and/or epitopes in the PreS1 domain of surface antigen,could be combined into a single protein of two polypeptide chains andtwo specificities, and thus greater affinity. Alternatively, an antibodytargeting HBV surface antigen and an antibody targeting CD3 can beexpressed in the liver, generating Fc dimerization and generation of amixture anti-HBsAg antibody, anti-CD3 antibody, and bispecificanti-HBsAg/CD3-Fc antibodies. Mutations in the Fc domains (ex:knob-in-hole designs) can facilitate more heterologous Fc chain pairing.

The disclosure further encompasses co-administration protocols withother compounds, e.g. bispecific antibody constructs, targeted toxins orother compounds, which act via immune cells, although in specific casesthe additional therapy does not act via immune cells. The clinicalregimen for co-administration of the inventive compound(s) may encompasscoadministration at the same time, before and/or after theadministration of the other component. Particular combination therapiesinclude reverse transcriptase inhibitors targeting HBV (example:lamivudine, adefovir, dipivoxil, telbivudine, tenofovir alafenamide,tenofovir, and entecavir); Interferon alfa-2b, pegylated interferon,chemotherapy, radiation, surgery, hormone therapy, arterialembolization, or other types of immunotherapy. In certain embodiments,compositions of the disclosure are utilized in liver transplant, forexample with administration to the liver graft prior to transplantation,in the cadaveric donor, perfused into the excised organ, and/or given tothe liver recipient.

Embodiments relate to a kit comprising one or more polynucleotides asdescribed herein, one or more polypeptides as described herein, and/or avector as described herein. It is also contemplated that the kit of thisdisclosure comprises a pharmaceutical composition as described herein,either alone or in combination with further medicaments to beadministered to an individual in need of medical treatment orintervention.

The polynucleotide introduction need not result in integration in mostcases. In many situations, transient maintenance of the polynucleotideintroduced may be sufficient. In this way, one could have a short termeffect, where gene vector could be introduced into the host at a localsite or organ and after introduction, then turned off, for example,after inflammation has been induced at a particular site. In the case ofmRNA modality for protein expression, the natural half-life of themolecule limits expression to 48-72 hours at most, in some embodiments.

It should be appreciated that the system is subject to many variables,such as the cellular response to the ligand, the efficiency ofexpression and, as appropriate, the level of secretion, the activity ofthe expression product, the particular need of the patient, which mayvary with time and circumstances, the rate of loss of the cellularactivity as a result of loss of cells or expression activity ofindividual cells, and the like. Therefore, it is expected that for eachindividual patient each patient would be monitored for the proper dosagefor the individual, and such practices of monitoring a patient areroutine in the art. Furthermore, monitoring of tissue damage andtoxicities is also envisioned, such as alanine aminotransferase (ALT)and aspartate aminotransferase (AST) measurements for liver toxicity,and are routine clinical measurements well known in the art.

III. Pharmaceutical Compositions

In accordance with this disclosure, the term “pharmaceuticalcomposition” relates to a composition for administration to anindividual. In specific aspects of the disclosure, the pharmaceuticalcomposition comprises a polynucleotide that encodes a polypeptide thatcomprises at least one immunostimulatory entity and/or at least oneliver antigen-targeting entity and/or the encoded polypeptide thereof.In a particular embodiment, the pharmaceutical composition comprises acomposition for parenteral, transdermal, intraluminal, intra-arterial,intrathecal or intravenous administration or for direct injection into acancer. It is in particular envisaged that the pharmaceuticalcomposition is administered to the individual via infusion or injection.Administration of the suitable compositions may be effected by differentways, e.g., by intravenous, subcutaneous, intraperitoneal,intramuscular, topical or intradermal administration, and in alternativeembodiments it occurs by infusion (such as catheter-based infusion),such as hepatic artery or portal vein infusion. In specific embodimentsfor hepatic artery infusion, the infusion is not constant; the infusionmay occur 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more times, inspecific embodiments, including within a specific and suitable timeframe.

The pharmaceutical composition of the present disclosure may furthercomprise a pharmaceutically acceptable carrier. Examples of suitablepharmaceutical carriers are well known in the art and include phosphatebuffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions, etc.Compositions comprising such carriers can be formulated by well-knownconventional methods. These pharmaceutical compositions can beadministered to the subject at a suitable dose.

The dosage regimen will be determined by the attending physician andclinical factors. As is well known in the medical arts, dosages for anyone patient depends upon many factors, including the patient's size,body surface area, age, the particular compound to be administered, sex,time and route of administration, general health, and other drugs beingadministered concurrently. Progress can be monitored by periodicassessment.

The compositions of the disclosure may be administered locally, althoughin alternative embodiments it is administered systemically, so long asit does not elicit harmful side effects. Administration may generally beparenteral, e.g., intravenous; DNA may also be administered directly tothe target site, e.g., by biolistic delivery to an internal or externaltarget site or by catheter to a site in an artery. In one embodiment,the pharmaceutical composition is administered subcutaneously and inanother embodiment intravenously. Preparations for parenteraladministration include sterile aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's, or fixed oils. Intravenous vehicles include fluid andnutrient replenishes, electrolyte replenishers (such as those based onRinger's dextrose), and the like. Preservatives and other additives mayalso be present such as, for example, antimicrobials, anti-oxidants,chelating agents, and inert gases and the like. In addition, thepharmaceutical composition of the present disclosure might compriseproteinaceous carriers, like, e.g., serum albumin or immunoglobulin,preferably of human origin. It is envisaged that the pharmaceuticalcomposition of the disclosure might comprise, in addition to theconstructs or nucleic acid molecules or vectors encoding the same (asdescribed in this disclosure), further biologically active agents,depending on the intended use of the pharmaceutical composition.

The dosing amounts may follow closely with previously establishedclinical parameters in humans and primates for achieving hightransduction of hepatocytes. An example of AAV gene therapy vectordosing may be 1 to 9×1012 vector genomes/kg intravenous infusion fortargeting to the liver. For mRNA delivery by lipid nanoparticle to theliver, an example of a dose of 0.025 mg/kg to 0.250 mg/kg mRNA perinjection can be infused intravenously in nanoparticles for efficientdelivery to the majority of hepatocytes. Multiple dosing cycles areenvisioned as necessary to fulfill therapeutic efficacy.

IV. Lipid Formulations

In particular embodiments of the disclosure, one or more compositionsare formulated in a lipid formulation. In specific embodiments, alipid-based nanoparticle is employed for one or more compositions. Inparticular cases, a vector comprising nucleic acid that encodes acomposition of interest, such as the nucleic acid being messenger RNA,may be delivered in a lipid-based nanoparticle.

In some embodiments, according to the present invention, a lipidsolution contains a mixture of lipids suitable to form lipidnanoparticles for encapsulation of mRNA. In some embodiments, a suitablelipid solution is ethanol based. For example, a suitable lipid solutionmay contain a mixture of desired lipids dissolved in pure ethanol (i.e.,100% ethanol). In another embodiment, a suitable lipid solution isisopropyl alcohol based. In another embodiment, a suitable lipidsolution is dimethylsulfoxide-based. In another embodiment, a suitablelipid solution is a mixture of suitable solvents including, but notlimited to, ethanol, isopropyl alcohol and dimethylsulfoxide.

A suitable lipid solution may contain a mixture of desired lipids atvarious concentrations. For example, a suitable lipid solution maycontain a mixture of desired lipids at a total concentration of orgreater than about 0.1 mg/ml, 0.5 mg/ml, 1.0 mg/ml, 2.0 mg/ml, 3.0mg/ml, 4.0 mg/ml, 5.0 mg/ml, 6.0 mg/ml, 7.0 mg/ml, 8.0 mg/ml, 9.0 mg/ml,10 mg/ml, 15 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, or 100mg/ml. In some embodiments, a suitable lipid solution may contain amixture of desired lipids at a total concentration ranging from about0.1-100 mg/ml, 0.5-90 mg/ml, 1.0-80 mg/ml, 1.0-70 mg/ml, 1.0-60 mg/ml,1.0-50 mg/ml, 1.0-40 mg/ml, 1.0-30 mg/ml, 1.0-20 mg/ml, 1.0-15 mg/ml,1.0-10 mg/ml, 1.0-9 mg/ml, 1.0-8 mg/ml, 1.0-7 mg/ml, 1.0-6 mg/ml, or1.0-5 mg/ml. In some embodiments, a suitable lipid solution may containa mixture of desired lipids at a total concentration up to about 100mg/ml, 90 mg/ml, 80 mg/ml, 70 mg/ml, 60 mg/ml, 50 mg/ml, 40 mg/ml, 30mg/ml, 20 mg/ml, or 10 mg/ml.

Any desired lipids may be mixed at any ratios suitable for encapsulatingmRNAs. In some embodiments, a suitable lipid solution contains a mixtureof desired lipids including cationic lipids, helper lipids (e.g. noncationic lipids and/or cholesterol lipids) and/or PEGylated lipids. Insome embodiments, a suitable lipid solution contain a mixture of desiredlipids including one or more cationic lipids, one or more helper lipids(e.g. non cationic lipids and/or cholesterol lipids) and one or morePEGylated lipids.

A. Cationic Lipids

As used herein, the phrase “cationic lipids” refers to any of a numberof lipid species that have a net positive charge at a selected pH, suchas physiological pH. Several cationic lipids have been described in theliterature, many of which are commercially available. Particularlysuitable cationic lipids for use in the compositions and methods of theinvention include those described in international patent publicationsWO 2010/053572 (and particularly, C12-200 described at paragraph[00225]) and WO 2012/170930, both of which are incorporated herein byreference.

In some embodiments, cationic lipids suitable for the compositions andmethods of the invention include a cationic lipid described in WO2015/184256 A2 entitled “Biodegradable lipids for delivery of nucleicacids” which is incorporated by reference herein such as3-(4-(bis(2-hydroxydodecyl)amino)butyl)-6-(4-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)butyl)-1,4-dioxane-2,5-dione (Target23),3-(5-(bis(2-hydroxydodecyl)amino)pentan-2-yl)-6-(5-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)pentan-2-yl)-1,4-dioxane-2,5-dione(Target 24).

In some embodiments, cationic lipids suitable for the compositions andmethods of the invention include a cationic lipid described in WO2013/063468 and in U.S. provisional application entitled “LipidFormulations for Delivery of Messenger RNA”, both of which areincorporated by reference herein. In some embodiments, a cationic lipidcomprises a compound of formula I c1-a:

-   -   or a pharmaceutically acceptable salt thereof, wherein:    -   each R₂ independently is hydrogen or CI 3 alkyl;    -   each q independently is 2 to 6;    -   each R′ independently is hydrogen or CI 3 alkyl;    -   and each RL independently is C8 12 alkyl.

In some embodiments, each R₂ independently is hydrogen, methyl or ethyl.In some embodiments, each R₂ independently is hydrogen or methyl. Insome embodiments, each R2 is hydrogen.

In some embodiments, each q independently is 3 to 6. In someembodiments, each q independently is 3 to 5. In some embodiments, each qis 4.

In some embodiments, each R′ independently is hydrogen, methyl or ethyl.In some embodiments, each R′ independently is hydrogen or methyl. Insome embodiments, each R independently is hydrogen.

In some embodiments, each RL independently is C8 12 alkyl. In someembodiments, each RL independently is n-C8 12 alkyl. In someembodiments, each RL independently is C9 11 alkyl. In some embodiments,each RL independently is n-C9 11 alkyl. In some embodiments, each RLindependently is C10 alkyl. In some embodiments, each RL independentlyis n-C10 alkyl.

In some embodiments, each R₂ independently is hydrogen or methyl; each qindependently is 3 to 5; each R independently is hydrogen or methyl; andeach RL independently is C8 12 alkyl.

In some embodiments, each R₂ is hydrogen; each q independently is 3 to5; each R is hydrogen; and each RL independently is C8 12 alkyl.

In some embodiments, each R₂ is hydrogen; each q is 4; each R ishydrogen; and each RL independently is C8 12 alkyl.

In some embodiments, a cationic lipid comprises a compound of formula Ig:

or a pharmaceutically acceptable salt thereof, wherein each RLindependently is C8 12 alkyl. In some embodiments, each RL independentlyis n-C8 12 alkyl. In some embodiments, each RL independently is C9 11alkyl. In some embodiments, each RL independently is n-C9 11 alkyl. Insome embodiments, each RL independently is C10 alkyl. In someembodiments, each RL is n-C10 alkyl.

In particular embodiments, a suitable cationic lipid is cKK-E12, or(3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione).Structure of cKK-E12 is shown below:

Additional exemplary cationic lipids include those of formula I:

-   -   and pharmaceutically acceptable salts thereof,    -   wherein,    -   R is

-   -   R is

-   -   R is

-   -    or    -   R is

(see, e.g., Fenton, Owen S., et al. “Bioinspired Alkenyl Amino AlcoholIonizable Lipid Materials for Highly Potent In vivo mRNA Delivery.”Advanced materials (2016)).

In some embodiments, one or more cationic lipids suitable for thepresent invention may beN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or“DOTMA”. (Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S.Pat. No. 4,897,355). Other suitable cationic lipids include, forexample, 5-carboxyspermylglycinedioctadecylamide or “DOGS,”2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumor “DOSPA” (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989); U.S.Pat. Nos. 5,171,678; 5,334,761), 1,2-Dioleoyl-3-Dimethylammonium-Propaneor “DODAP”, 1,2-Dioleoyl-3-Trimethylammonium-Propane or “DOTAP”.

Additional exemplary cationic lipids also include1,2-distearyloxy-N,N-dimethyl-3-aminopropane or “DSDMA”,1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or “DODMA”,1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or “DLinDMA”,1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or “DLenDMA”,N-dioleyl-N,N-dimethylammonium chloride or “DODAC”,N,N-distearyl-N,N-dimethylarnrnonium bromide or “DDAB”,N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide or “DMRIE”,3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propaneor “CLinDMA”, 2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethy1-1-(cis,cis-9′, 1-2′-octadecadienoxy)propane or “CpLinDMA”,N,N-dimethyl-3,4-dioleyloxybenzylamine or “DMOB A”,1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane or “DOcarbDAP”,2,3-Dilinoleoyloxy-N,N-dimethylpropylamine or “DLinDAP”,1,2-N,N-Dilinoleylcarbamyl-3-dimethylaminopropane or “DLincarbDAP”,1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane or “DLinCDAP”,2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane or “DLin- -DMA”,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or“DLin-K-XTC2-DMA”, and 2-(2,2-di((9Z, 12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLin-KC2-DMA))(see, WO 2010/042877; Semple et al., Nature Biotech. 28: 172-176(2010)), or mixtures thereof. (Heyes, J., et al., J Controlled Release107: 276-287 (2005); Morrissey, D V., et al., Nat. Biotechnol. 23(8):1003-1007 (2005); PCT Publication WO2005/121348A1). In some embodiments,one or more of the cationic lipids comprise at least one of animidazole, dialkylamino, or guanidinium moiety.

In some embodiments, one or more cationic lipids may be chosen from XTC(2,2-Dilinoley 1-4-dimethylaminoethy 1-[1,3]-dioxolane), MC3(((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate), ALNY-100((3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z, 12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)), NC98-5(4,7, 13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7, 10,13-tetraazahexadecane-1, 16-diamide), DODAP(1,2-dioleyl-3-dimethylammonium propane), HGT4003 (WO 2012/170889, theteachings of which are incorporated herein by reference in theirentirety), ICE (WO 2011/068810, the teachings of which are incorporatedherein by reference in their entirety), HGT5000 (U.S. Provisional PatentApplication No. 61/617,468, the teachings of which are incorporatedherein by reference in their entirety) or HGT5001 (cis or trans)(Provisional Patent Application No. 61/617,468), aminoalcohol lipidoidssuch as those disclosed in WO2010/053572, DOTAP(1,2-dioleyl-3-trimethylammonium propane), DOTMA(1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (Heyes, J.;Palmer, L.; Bremner, K.; MacLachlan, I. “Cationic lipid saturationinfluences intracellular delivery of encapsulated nucleic acids” J.Contr. Rel. 2005, 107, 276-287), DLin-KC2-DMA (Semple, S.C. et al.“Rational Design of Cationic Lipids for siRNA Delivery” Nature Biotech.2010, 28, 172-176), C12-200 (Love, K. T. et al. “Lipid-like materialsfor low-dose in vivo gene silencing” PNAS 2010, 107, 1864-1869).

In some embodiments, cationic lipids constitute at least about 5%, 10%,20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the total lipidsin a suitable lipid solution by weight or by molar. In some embodiments,cationic lipid(s) constitute(s) about 30-70% (e.g., about 30-65%, about30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%), about35-50%), about 35-45%, or about 35-40%) of the total lipid mixture byweight or by molar.

B. Non-cationic/Helper Lipids

As used herein, the phrase “non-cationic lipid” refers to any neutral,zwitterionic or anionic lipid. As used herein, the phrase “anioniclipid” refers to any of a number of lipid species that carry a netnegative charge at a selected pH, such as physiological pH. Non-cationiclipids include, but are not limited to, distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monom ethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or a mixturethereof.

In some embodiments, non-cationic lipids may constitute at least about5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% ofthe total lipids in a suitable lipid solution by weight or by molar. Insome embodiments, non-cationic lipid(s) constitute(s) about 30-50%(e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about35-40%)) of the total lipids in a suitable lipid solution by weight orby molar.

C. Cholesterol-based Lipids

In some embodiments, a suitable lipid solution includes one or morecholesterol-based lipids. For example, suitable cholesterol-basedcationic lipids include, for example, DC-Choi(N,N-dimethyl-N-ethylcarboxamidocholesterol),1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys.Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997);U.S. Pat. No. 5,744,335), or ICE. In some embodiments, cholesterol-basedlipid(s) constitute(s) at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%,or 70%) of the total lipids in a suitable lipid solution by weight or bymolar. In some embodiments, cholesterol-based lipid(s) constitute(s)about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%), about35-45%, or about 35-40%) of the total lipids in a suitable lipidsolution by weight or by molar.

D. PEGylated Lipids

In some embodiments, a suitable lipid solution includes one or morePEGylated lipids. For example, the use of polyethylene glycol(PEG)-modified phospholipids and derivatized lipids such as derivatizedceramides (PEG-CER), includingN-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol)-2000](C8 PEG-2000 ceramide) is also contemplated by the present invention.Contemplated PEG-modified lipids include, but are not limited to, apolyethylene glycol chain of up to 5 kDa in length covalently attachedto a lipid with alkyl chain(s) of C6-C20 length. In some embodiments, aPEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. Insome embodiments, particularly useful exchangeable lipids arePEG-ceramides having shorter acyl chains (e.g., C14 or C18).

PEG-modified phospholipid and derivatized lipids may constitute at leastabout 5%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the total lipids in asuitable lipid solution by weight or by molar. In some embodiments,PEGylated lipid lipid(s) constitute(s) about 30-50% (e.g., about 30-45%,about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the totallipids in a suitable lipid solution by weight or by molar.

Exemplary combinations of cationic lipids, non-cationic lipids,cholesterol-based lipids, and PEG-modified lipids are described in theExamples section. For example, a suitable lipid solution may containcKK-E12, DOPE, chol, and DMG-PEG2K; C12-200, DOPE, cholesterol, andDMG-PEG2K; HGT5000, DOPE, chol, and DMG-PEG2K; HGT5001, DOPE, chol, andDMG-PEG2K; cKK-E12, DPPC, chol, and DMG-PEG2K; C12-200, DPPC,cholesterol, and DMG-PEG2K; HGT5000, DPPC, chol, and DMG-PEG2K; orHGT5001, DPPC, chol, and DMG-PEG2K. The selection of cationic lipids,non-cationic lipids and/or PEG-modified lipids which comprise the lipidmixture as well as the relative molar ratio of such lipids to eachother, is based upon the characteristics of the selected lipid(s) andthe nature of the and the characteristics of the mRNA to beencapsulated. Additional considerations include, for example, thesaturation of the alkyl chain, as well as the size, charge, pH, pKa,fusogenicity and toxicity of the selected lipid(s). Thus the molarratios may be adjusted accordingly.

V. Kits of the Disclosure

Any of the compositions described herein, or components thereof, may becomprised in a kit. In a non-limiting example, polynucleotides, cells orany reagents to manipulate or generate certain polynucleotides,proteins, peptides and/or cells may be comprised in a kit. Such a kitmay or may not have one or more reagents for manipulation of molecules.Such reagents may include small molecules, proteins, nucleic acids,antibodies, buffers, primers, nucleotides, salts, and/or a combinationthereof, for example. In particular embodiments, a polynucleotide thatencodes a polypeptide that comprises a liver antigen-targeting entityand/or an immunostimulatory entity, or each component separately, orprimers suitable for amplifying either entity, may be provided in a kit.In some cases, cells for harboring such a polynucleotide(s) may beprovided in a kit, and/or an apparatus to obtain cells from anindividual may be provided in the kit. The kit may have one or morereagents tailored to a particular one or more liver antigens and/or oneor more immunostimulatory entities.

In particular aspects, the kit comprises the polynucleotide and/orpolypeptide therapy of the disclosure and also another therapy. In somecases, the kit, in addition to the polynucleotide and/or polypeptidetherapy embodiments and wherein the individual has cancer, also includesa second cancer therapy, such as chemotherapy, hormone therapy, and/orimmunotherapy, for example. The kit(s) may be tailored to a particularcancer for an individual and comprise respective second cancer therapiesfor the individual.

The kits may comprise suitably aliquoted compositions of the presentinvention. The components of the kits may be packaged either in aqueousmedia or in lyophilized form. The container means of the kits willgenerally include at least one vial, test tube, flask, bottle, syringeor other container means, into which a component may be placed, andpreferably, suitably aliquoted. Where there are more than one componentin the kit, the kit also may generally contain a second, third or otheradditional container into which the additional components may beseparately placed. However, various combinations of components may becomprised in a vial. The kits of the present invention also willtypically include a means for containing the composition and any otherreagent containers in close confinement for commercial sale. Suchcontainers may include injection or blow-molded plastic containers intowhich the desired vials are retained.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. In which case, thecontainer means may itself be a syringe, pipette, and/or other such likeapparatus, from which the formulation may be applied to an infected areaof the body, injected into an animal, and/or even applied to and/ormixed with the other components of the kit. However, the components ofthe kit may be provided as dried powder(s). When reagents and/orcomponents are provided as a dry powder, the powder can be reconstitutedby the addition of a suitable solvent. It is envisioned that the solventmay also be provided in another container means.

EXAMPLES

The following examples are presented in order to more fully illustratethe preferred embodiments of the disclosure. They should in no way,however, be construed as limiting the broad scope of the disclosure.

Example 1

A cartoon schematic of a single bispecific antibody design isillustrated in FIG. 1 . In this example, the n-terminal end possesses anscFv XTL19 or 19 targeting HBsAg, the linker region is an Fc domainderived from human IgG1, and the c-terminal domain harbors an scFvagainst mouse CD3, in order to facilitate testing in mouse models. Therecombinant protein would then be delivered for expression by genetherapy into the liver. An example of a mechanism for how suchbispecific molecules function is shown in FIG. 2 , wherein the moleculesconnect an antigen target (here HBsAg on cell surface) to T cells viaCD3 binding (could be other cell types in other embodiments) leading tothe clustering of CD3 molecules together resulting in T cell signalingand activation. In the testing system in mouse models, the inventorsemployed co-delivery of plasmid encoding target (HBV) plus luciferasereporter (see FIG. 3 for an example) and a plasmid encoding thebispecific antibody therapy under a CAG promoter are provided thatsimulates gene therapy into a human patient into the liver organ andlimited expression in other tissues. The target antigen is onlyexpressed in the liver along with the bispecific molecule, andluciferase mediated luminescence provides a convenient real-time readoutfor immune reaction inside the liver.

In an acute model of HBV infection, the artificial T cell responseagainst HBV removed to roughly 100-fold the genome levels, compared tothose of an early time point where no immune response against virus hasstarted (day 4 and day 8) (FIG. 4 ). Improvement was also noted over theefficacy of antibody targeting antigen alone (19-Fc). This early settingsimulates the human patient having no immune response to the virus atbaseline. Current therapies do not target viral genomes, and genomedecline (HBV covalently closed circular DNA) is marginal (2-fold) withRT inhibitors. There was observed a dose response to the efficacy ofknockdown, indicating the centrality of T cell activation in thesuppression and removal of HBV. More gene delivered to the liverresulted in a saturating dose at 15 μg and 20 μg levels (FIG. 5 ).

FIG. 6 demonstrates the visualization of the quantitative data. Similarexperiments in tumor models are the gold standard for measuringresponses. The drastic reduction in an immunocompetent animal modelrelying on recruitment of local T cells in the liver indicates that theapproach is directly applicable to human therapy. Results representcomplete viral clearance in a week, while current therapies in patientsdo not clear the viral genomes, but merely suppress serum markers.

Experiment 2 Hepatic Gene Therapy Expressing Bispecific AntibodiesRedirects T Cells to Mediate Potent Antiviral Responses AgainstHepatitis B Virus

Novel therapies against hepatitis B virus (HBV) are needed to cure virusfrom patients, which cannot currently be achieved by drugs today. T cellresponses clear HBV in acute infection, and adoptive transfer ofantiviral T cells can lead to significant reductions in vivo. Seekingmore scalable methods to harness T cells against HBV, the inventorsdeveloped a novel method of activating host T cells in situ in the liverfor HBV therapy. Genes for bispecific antibodies binding to HBsAg andCD3 epsilon were delivered directly into the liver by hydrodynamic tailvein injection, where after they found murine T cells mediated multi-logreduction in HBsAg and reporter gene expression within 1 day. In situexpressed bispecific antibodies were prone to antigen-independent T cellactivation in the liver microenvironment, affording resistance topotential viral mutation escape. This was a novel an unexpected finding,because traditionally bispecific antibodies do not activate immune cellsin the absence of target antigen. In addition, in situ bispecificantibody production was not cytotoxic to hepatocytes, and the antiviraleffect was largely noncytopathic. Finally, bispecific antibodiespotently activated host anti-HBsAg antibody production after theirexpression, suggesting additional potential as an in situ vaccine.Overall, this strategy is useful for a clinical therapy for chronic HBVinfection.

Introduction: Hepatitis B virus (HBV) currently chronically infects over300 million people today. There is currently no cure for theseindividuals who are chronically infected. While current drugs cansuppress serum HBV DNA levels, there is no effect on covalently closedcircular DNA (cccDNA), the viral genome of HBV. In particular, thehepatitis B surface antigen (HBsAg) production is not curtailed, a keymolecule that is thought to suppress the immune response, possiblythrough inhibiting plasmacytoid dendritic cells (Xu, et al., 2009) andinnate immune signaling (Liu, et al., 2015). Newer proposed therapiesdegrade HBV RNA's using siRNA (Wooddell, et al., 2013) and antisensemodalities (Billioud, et al., 2016), effectively knocking down HBsAgexpression. However, HBV cccDNA remains untouched. It is hypothesizedthat HBV could be cured through re-activating the immune system to clearcccDNA by relieving the HBsAg-mediated suppression (Durantel & Zoulim,2016), but this remains to be realized in human trials since preclinicalanimals models can't adequately test it.

More direct approaches of activating the immune system against HBV couldprove to be an effective therapy toward cure. The CD8 T cell response iscrucial toward clearing HBV in the liver (Thimme, et al., 2003).Furthermore, clearance is largely noncytopathic, relying primarily onsecreted cytokines, INF-γ and TNF-α (Xia, et al., 2016). However, in HBVpatients, the frequency of HBV-specific T cells is low (Boni, et al.,2007) and their functionality is impaired (Park, et al., 2016). Vaccinestrategies depend on activating T cells that might not be present, andHBV knockdown strategies relying on a potentially dysfunctional immunesystem might prove to be fruitless.

T cells were previously redirected to attack HBV infected hepatocytesusing chimeric antigen receptors (CAR) targeting HBsAg (Bohne, et al.,2008). While primarily secreted, there is a residual amount of HBsAgdetected on the surface of infected hepatocytes that is recognizable byCAR-T cells. Redirected T cells were shown to reduce cccDNA frominfected primary hepatocytes in vitro, and mediate transient viralreduction in an HBV transgenic mouse model (Krebs, et al., 2013).

While CAR-T cells represent a potential tool against HBV, the largenumber of HBV patients worldwide demand a more readily available off theshelf strategy. Toward this goal, the use of bispecific antibodiesagainst HBV was investigated, which could give an adaptive immuneresponse back to HBV through providing both humoral and cellularimmunity. In particular, in an effort to directly activate host T cellstoward given HBV-infected cells, bispecific antibodies can beconstructed that targets both HBsAg on the surface of hepatocytes andCD3 epsilon (CD3) on the surface of T cells. Bispecific antibodiestargeting CD3 were originally reported over 30 years ago (Staerz, etal., 1985; Staerz, et al., 1986) and have been seen in numerousapplications since then, including most recently the FDA approvedblinatumomab targeting the CD 19 antigen (Przepiorka, et al., 2015).These molecules work by binding a target antigen on the cell surface,where after binding that antigen causes clustering of CD3 proteins onthe T cell surface, triggering activation similar to the TCR complex.The advantage of this strategy is MHC independent T cell activation,facilitating the opportunity for general off the shelf strategies forall patients.

Current bispecific antibody approaches are challenged by complicatedmanufacturing process, complex pharmacokinetics requiring constantinfusion, and potential toxicity issues through systemic T cellactivation. All of these hurdles could be addressed through in situexpression of bispecific antibodies from DNA or RNA templates in patienttissues directly, but such attempts to express these genes directly intissues have not been reported in the literature, but rather focused onsecretion by cell vehicles (Compte, et al., 2013). Herein, there is abispecific antibody against HBV, that when delivered to the livertissue, mediates rapid reduction of the virus in an immunocompetentmouse model.

Examples of Results

A gene therapy strategy was selected toward delivering the bispecificantibodies into the patient for multiple reasons: 1) soluble HBsAg inthe serum will readily neutralize a majority of the infused therapeutic;2) risk that T cells are systemically activated via cross-linking of CD3induced by soluble surface antigen particles; 3) in clinical trials withHBV antibodies, it has been noted that a quick metabolism of antibodiestakes place through the aforementioned endocytosis into the liver, suchthat decrease only last hours (Neumann, et al., 2010); 4) in clinicaltrials for bispecific T cell therapies, a continuous infusion ofantibodies is often needed in order to measure a therapeutic effect(Ribera, et al., 2015), likely due to the logistics of the needed tosimultaneously engage two different cells via a single molecule; and 5)reports of immune-complex disorders have occurred when anti-HBsAgantibodies have been infused into HBV patients (van Nunen, et al.,2001). Through a gene therapy strategy then, HBV immunity could be givento patients directly at the site of the liver and activate T cells in aspecific and safe manner.

Given the novel focus on designing bispecific antibody therapies to beexpressed in patient tissue, it was considered that optimizingartificial tissue culture conditions with varying amounts of T cells,producer cells, and target cells would not be informative for the invivo context. The liver represents a complex microenvironment andarchitecture of different cells and components. T cells in circulationmust reach through gaps in endothelial cells forming the space of Disseto even reach the hepatocyte membrane (Guidotti, et al., 2015), makingit unclear if secreted molecules to effectively bridge these twotargets. For all these reasons, direct tests in preclinical mouse modelsystems were selected.

It was desired to assess the treatment modality in a setting close tohuman infection and to use an immunocompetent mouse model, to allow forthe recruitment of host T cells to HBV infected hepatocytes in vivo.This would include within their natural environment a more authenticratio of T cells to hepatocytes that one might expect in the humanpatient, along with containing all the other important cell players suchas Kupffer cells and natural killer cells, that could play additionalroles in this therapy. Furthermore, one could follow subsequent adaptiveimmune responses in the mouse to the intervention.

In order to deliver both HBV and antibody genes into murine liver,hydrodynamic tail vein injection was employed (Liu, et al., 1999). Thehydrodynamic model of HBV infection has previously been demonstrated tobe convenient model of HBV via direct delivery of HBV plasmids, normallyresulting in acute infection in most strains (Yang, et al., 2002; Chen,et al., 2012). Furthermore, this method can also be used to introducetherapeutics genes at into the liver (Viecelli, et al., 2014)′ Alino, etal., 2003), which in this case will be co-injected bispecific antibodygenes.

Antibody Production by Hepatocytes and HBV Luminescence Model Validation

It was desired first to establish the ability to delivery antibodyproteins into the mouse liver for their expression in situ, resulting intheir correct expression and secretion, while retaining their affinityfor HBsAg. For these studies, the inventors chose to focus on a humanantibody, mAb19.79.5 of XTL Pharma (known as XTL19 or 19 herein),previously validated in preclinical models (Eren, et al., 2000) and inclinical trials (Galun, et al., 2002) and effectively neutralizes HBVvirions. The antibody targets a linear epitope on the “a” determinant ofthe small HBsAg with nanomolar affinity (Eren, et al., 1998).Hydrodynamic tail vein injection was carried out at 2 different doses (5ug, 15 ug plasmid) of the hybrid antibody 19-Fc, consisting of thesingle chain variable fragment (scFv) of the XTL 19 antibody cloneappended to the Fc domain of the human IgG1 protein (FIG. 7A). Antibodyexpression was driven under the CMV early enhancer/chicken β actin (CAG)hybrid promoter that has been previously noted for high expressionpotency and resistance to silencing in the murine liver (Nguyen, et al.,2008), as opposed to CMV driven constructs (Kay, et al., 1992). The twodifferent doses achieved serum anti-HBsAg antibody levels of 8 and 16mlU/mL at day 4 post injection, with the 15 ug dose yielding antibodylevels sufficient to protect a human patient from HBV infection(McMahon, et al., 2009) (FIG. 7B). It is likely that the effectiveantibody concentration within the liver microenvironment is much higherthan the overall concentration in the serum, increasing potentialpotency.

With the ability to produce antibodies in hepatocytes verified, theinventors next sought to establish a system for monitoring therapeuticefficacy against virus in vivo in realtime, with a focus on the viralgenomic stability given that targeting cccDNA is an important aspect fora sterilizing cure. Rather than focusing on serum markers, which mayfluctuate but not inform on the levels of actual plasmid genome insidethe cells, a bioluminescence system using firefly luciferase was used. Aplasmid, HBV-Luc, was constructed harboring the HBV 1.3 overlengthgenome with the second HBV core promoter driving a fusion protein ofGFP-2A-luciferase (FIG. 7C). The sequence of this construct is given inFIG. 13 . Luciferase expression can be monitored in vivo as a readout ofthe immune response, as previously demonstrated in similar systems forHBV (Liang, et al., 2013) and malaria (Rai, et al., 2012). Furthermore,the HBV study found luminescence is a directly correlated to serum HBsAgand HBV DNA levels (Liang, et al., 2013). On a direct level, plasmidlevels and HBV core promoter activity in the liver are tied toluminescence. The 5 ug HBV-Luc plasmid could successfully expressluciferase after hydrodynamic injection (FIG. 7D), and could beexpressed long-term in NOD SCID−/−γ−/−(NSG) mice in the absence of anadaptive immune response (FIG. 7E). This validates its use going forwardin testing HBV therapeutic interventions.

In Vivo Screening for Bispecific Antibody Format Efficacy

There are multiple bispecific antibody formats with different geometriesand valencies in the literature (Wei die, et al., 2012; Spiess, et al.,2015), and the inventors wanted to carry out a screening approach toascertain the most potent protein in the context of hepatocyte-mediatedproduction. Since the goal is to activate T cells in mice, an scFv wasselected encoding the hamster 145-2C11 antibody clone binding to themouse CD3 epsilon (mCD3) (Leo, et al., 1987), since engagement of thisprotein can activate murine T cells selectively. Furthermore, the scFvhas been utilized in previous studies in order to construct bispecificantibodies to redirect murine T cells (Jost, et al., 1996). It was alsotested whether adding costimulation with murine CD80 or B7.1 ectodomaincould help engage CD28 receptors, as an alternative activation domain(Haile, et al., 2013). The inventors also tested one format with theseactivation moieties directly linked to the XTL19 scFv similar to thebispecific T cell engager (BiTE) format, along with a format includingall of mCD3, mB7.1, and XTL19.

It is known that HBV antibodies have the ability to both neutralizeinfection and mediate faster clearance of particles, but to also enterthe endosomes of hepatocytes and block the secretion of HBsAg particles(Schilin, et al., 2013). This function could help an inherent challengein bispecific targeting, wherein the soluble HBsAg particles distractantibodies away from the target cell surface. For one approach, it wasconsidered that engineering the normal IgG antibody format for T cellengagement could solve both of these challenges. By connecting an scFvagainst HBV via an the IgG Fc to another scFv directed against CD3epsilon, one could both effectively neutralize HBV, block HBsAg, andrecruit T cells to the surface of infected hepatocytes.

It was also considered whether or not to keep Fc receptor binding inthese antibodies, which could add additional potency but alsotoxicities. The inventors desired to keep the function of neonatal Fcreceptor (FcRn) binding, which is crucial for the inhibition of FIB sAgsecretion as demonstrated in previous studies (Schilling, et al., 2003).Towards safety, the other effector functions might cause potentialtoxicities. In a clinical trial, antibody-complex toxicities were notedin patients treated with anti-HBsAg antibodies (van Nunen, et al.,2001). Furthermore, in clinical trials with the bispecific catumaxomaband wildtype Fc domain, toxicity was noted during intravenous injection,via binding and cross-linking with FcR's causing systemic T cellactivation (Mau-Sorensen, et al., 2015). In order to resolve this issue,it was decided to use IgG4 Fc domain with mutations in the linker regionand in the CH2 domain that abrogate Fc receptor binding (Hudecek, etal., 2015). Another bispecific version had additional mutations to makethe Fc domain monomeric in structure (Ying, et al., 2012). Furthermore,it was considered that some toxicity could be avoided with localizeddelivery of antibody to tissue site, and reduced CD3 affinity with scFvlocalization at c-terminus (Kuo, et al., 2012).

The inventors thus tested both IgG1 Fc domains that could bind to Fcreceptors, and an IgG4 Fc domain with mutations to abrogate Fc receptorbinding (Hudecek, et al., 2015). There was testing of appendage of themCD3 scFv onto either the n-terminus or c-terminus of the Fc domain,with the XTL19 scFv occupying the other site. mB7.1 was added to then-terminus in another format to provide costimulation signals toT-cells. A summary of the antibodies tested can be found in FIG. 8A.These various formats were systematically tested by co-injecting 15 ugAntibody with 5 ug HBV-Luc. As a control, 15 ug CMV-Gaussia wasco-injected, being also a secreted protein but having no activatingeffect on the immune system. A positive control was also included ofmCD3 antibody with inert Fc known to partially activate T cells (Smith,et al., 1997). At day 4, the peak of luciferase expression in thecontrol group, luminescence levels were decreased. Of note, all antibodyformats demonstrated efficacy against HBV, demonstrating the usefulnessof the different permutations in antibody design and immunostimulatorydomains that might be used in the current disclosure when delivered as agene therapy into the liver. 19-Fc-mCD3 and mCD3-19 exhibited the lowestdecreases in luminescence, and continued pursuing those in furtherstudies (FIG. 8B).

Treating Acute HBV Infection with Bispecific Antibodies

The inventors focused first on the 19-Fc-mCD3 bispecific, because thisformat could leverage the additional Fc functionality and have morepotential in therapeutic use, in at least certain aspects. This antibodywas tested in an acute HBV model in immunocompetent mice, wherehydrodynamic injection of HBV leads to viral clearance within weeks. Theresolution is characterized by the development of anti-HBsAg antibodiesand the infiltration of HBV-specific T cells into the liver (Yang, etal., 2002). It was considered that the bispecific will cause earlyreduction in HBV levels facilitating faster clearance in mice versuscontrol groups. A dosing study was undertaken in vivo with bispecificantibodies comparing to the injection with HBV alone, to find out thehighest dose response for therapy in this system. There was increasinginhibition of luciferase expression up to 15 ug CAG-19-Fc-mCD3, with 15and 20 ug being relatively the same (FIG. 3A). Therefore, the 15 ugplasmid dose were used going forward.

The potency of 19-Fc-mCD3 and its mechanism of action was tested. Therewas a 1.79 log luminescence knockdown that occurred with 19-Fc-mCD3targeted HBV at day 4 post injection (FIG. 9B). Notably, including themCD3 scFv at the c-terminus resulted in significantly more luminescencedecrease versus the 19-Fc antibody alone (0.82 log reduction, p<0.05).Furthermore, the Fc receptor function was not necessary for 19-Fc-mCD3function as a 19-G4m-mCD3 version had similar potency. Final clearancemay in part be driven by the presence of foreign protein, becauseGaussia inclusion facilitated faster clearance versus including an emptyplasmid construct at day 16. There was a 1.3-1.7 log decrease in HBsAglevels at day 4 with the 19-Fc and bispecific constructs, verifyingsimilar trends in luminescence data with a virological marker (FIG. 9C).

Antigen Independent T Cell Activation by Bispecific Antibodies in LiverMicroenvironment

With efficacy validated using bispecific constructs containing scFv 19and mCD3 binding, the specificity of the process was verified. A seriesof parallel antibodies were constructed with the XTL19 scFv domainreplaced with the EGFRvIII-specific 139 scFv. This antibody targets anepitope expressed in certain cancers, but not in normal murine tissue(Morgan, et al., 2012). A version was constructed with an scFv targetingEphA2 as another control (Iwahori, et al., 2015). Repeating the sameexperiment, the mCD3 containing 139-Fc-mCD3, 13-G4m-mCD3, EphA2-Fc-mCD3could all decrease luciferase levels, indicative of T cell activation(all p<0.05 vs CMV-Gaussia control) (FIG. 10A). By comparison, the139-Fc had no activity in decreasing luminescence versus control, asexpected (p=0.87). Similar antigen independent background activation hadbeen previously reported with this same bispecific design, albeitsignificantly less than the on target activation (Kuo, et al., 2012).The inventors also sought to compare two similar versions side by sidewith G4m domain linker, in order to remove the confounding role of Fcreceptors in facilitating antibody-T cell cross-linking. Looking atluminescence levels at Day 4, the 19-G4m-mCD3 construct decreasedluminescence significantly more than the 139-G4m-mCD3, suggesting thatthe 19 scFv might help trigger increased bispecific antibody clusteringand T cell activation in an antigen-dependent manner, as would beanticipated (FIG. 10B).

Because the Fc-containing antibodies inherently are dimeric, the twomCD3 binding portions alone might help facilitate some level of T cellactivation. It was considered whether monomeric bispecific moleculeswould have decreased or no antigen-independent T cell activation. Aseries of bispecific antibodies were constructed similar to the BiTEformat found in blinatumomab, which does not activate T cells in theabsence of target antigen (Brischwein, et al., 2007). The inventorsappended the mCD3 binding portion to the n-terminus of the BiTE sincethat has been reported to provide better efficacy in mouse models(Schlereth, et al., 2006). An scFv alone construct was also designedthat would bind mCD3 and lack any other binding component. Unexpectedly,the mCD3-139 and mCD3 alone vectors also stimulated T cell activationand luminescence decrease to similar levels as mCD3-19 (p<0.05 vsCAG-empty) (FIG. 10C). This suggests that the liver might readilyproduce aggregates of antibody proteins in over-expression conditionsthat could cause CD3 clustering, a common phenomenon during theindustrial recombinant protein production resolved in downstreamprocessing (Paul, et al., 2014), but cannot otherwise be removed in thissetting. Antigen-independent activation by bispecific antibodiessecreted from cell lines in vitro has previously been reported with thisstudy similar to that phenomenon (Compte, et al, 2014). The time courseof BiTE dependent activation was similar to the Fc containing constructspreviously studied, indicating no additional benefit in decreasingluminescence (FIG. 10D).

Mechanism of Action for Bispecific Antibodies in Clearing HBV

The mechanism of action of the bispecific antibodies was furtherconsidered. So far, antibody expression was driven with a CAG promoter,which after introduction could in certain embodiments lead to continuousproduction of antibody until all HBV is eliminated. On the other hand,the previous figures suggest that the effect might be only peak at day4, with the final elimination happening by plasmid inactivation or hostimmune response. To clarify, an experiment comparing bispecific activityin NSG and WT mice was performed. Bispecific antibodies in NSG miceshould be able to activate residual immature T cell progenitors (Falk,et al., 1996), and indeed the day 4 point between NSG and WT mice wassimilar (FIG. 11 A). After, however, the NSG mouse saw continuedpersistence of luminescence signal, while the WT mouse completelycleared, indicating that antibody is not continuously made through theexperiment and that host adaptive immunity is key for the later stagesof elimination on the curve. With the finding that bispecific antibodyaction happens early, the inventors wanted to find out how early.Similar experiments as FIG. 9 were repeated, except the luminescencesignal was measured every day for the first 4 days. A 1.03 logdifference between control and antibody treated already occurs at day 1post injection (FIG. 11B), with the similar difference largelymaintained subsequently. This suggests the first secreted bispecificmolecules must activate T cells on day 1, producing cytokines thatultimately help silence the plasmid CAG-antibody expression vector, inaddition to helping clear HB V.

It was desired to check if the expression of the antibodies themselvesby hepatocytes was toxic, which otherwise might produce the therapeuticeffects observed so far. The same experiments were repeated, butreplaced the HBV-Luc vector with a CMV-NLS-Cre plasmid. Plasmids wereco-injected into a ROSA-LoxP-STOP-LoxP Luciferase (Rosa-Luc) mousestrain, activate luciferase expression in the liver. The luminescencelevels were not significantly different between bispecific antibodyinjected and Gaussia injected mice at day 4 or day 8 (FIG. 11C),suggesting that the bispecific antibody production is not toxic tohepatocytes, whose death would have reduced signal.

The CRISPR-Cas9 system has the ability to specifically target and cutDNA sequences of choice, and has been proposed as a therapy to targetthe HBV DNA genome in numerous studies (Dong, et al., 2015; Seeger &Sohn, et al., 2014; Karimova, et al., 2015). On the other hand, it hasbeen reported recently that the mammalian immune systems have their ownability to up-regulate endogenous effects to degrade HBV cccDNA(Lucifora, et al., 2014). It was desired to compare the two approacheswith different mechanisms of action head to head in the same system, inorder to assess their merits. The inventors co-injected Cas9 withgRNA-21, previously found to be highly potent (Ramanan, et al., 2015),with HBV-Luc. CRISPR and the bispecific antibodies had similar knockdownat Day 4 (FIG. 11D), suggesting that inducing inflammation alone wouldbe an alternative to designer nuclease strategies.

Bispecific Antibody treatment of a cccDNA mouse model in vivo [00226]The ability of bispecific antibody therapy to induce cccDNA clearance invivo, representing a more authentic model for the human infection, wasinvestigated. To this purpose, the inventors utilized a tool comprisinga flox'd HBV genome with a LS-Cre (containing an internal intron)cassette driven by CMV promoter in cis on the same plasmid (FIG. 14 ).When this plasmid is introduced into Rosa-Luc mice, the Cre recombinaseactivates luciferase expression in the host cell chromosomes andepisomal cccDNA formation (FIG. 14 ). If HBV kills infected cells, thenthe luciferase signal should completely disappear with therapy. If onthe other hand, the bispecific antibody activates T cells to removecccDNA from mouse hepatocytes non-cytopathically, the luciferase signalshould remain at high levels, while HBV cccDNA is cleared from themouse. The inventors co-injected 15 ug of 19-G4m-mCD3, control139-G4m-mCD3, or Gaussia along with 5 ug Cre/LoxP-HBV (CLX) plasmid.Over time the two treated groups showed a loss in luminescence overtime, but that high levels of luminescence remained, indicating theoriginal infected cells were not entirely eliminated (FIG. 12A).Notably, the Gaussia treated group showed a much slower decline over thesame time course, suggesting some tolerance to HBV antigens. Because itwas established that the first days are when bispecific antibodiesprimarily function, in at least some embodiments, it was desired toassess the cytotoxicity at the first time point. The 19-G4m-mCD3 grouphad 75% less luminescence than the Gaussia control, which wasstatistically significant (FIG. 12B). At the same time point, the19-G4m-mCD3 group already had a 1.65 log drop in HBsAg levels,indicating that noncytopathic effects predominated, with cytotoxicitydriven by 19-G4m-mCD3 playing a minor role (FIG. 12C). While bispecificantibodies appear to only act briefly post injection, it was desired tosee their potential to modulate the adaptive immune response and act asan in situ vaccine. The development of anti-HBsAg antibodies in mice wasmeasured, and the 19-G4m-mCD3 group developed high titer antibodies atmuch faster and higher levels as compared to the 139-G4m-mCD3, whichlacked antigen binding, and the Gaussia group, which developed almost noantibodies during this period to HBsAg (FIG. 12D). This result suggeststhat that combination of antigen recognition and T cell activation,humoral and cell mediated arms, has significant advantages overactivation of T cells (139-G4m-mCD3 group) alone.

Given the importance of maintaining hepatocyte integrity and healthduring therapy, it was considered if one could co-deliver additionalproteins or molecules that could help protect hepatocytes fromdestruction in addition to the therapy. Bcl2 was previously shown toprevent Fas antibody induced fulminant hepatic failure in mice(Lacronique, et al. 1996). The concurrent question was if this mightinterrupt the ability of the bispecific antibody to mediate HBVsuppression. Mice (n=4+ were all hydrodynamically injected with 5 ugHBV-Luc plasmid, along with 5 ug CAG-Bcl2 or Control plasmid, plus 15 ug19-Fc-mCD3 plasmid. Expressing an anti-apoptotic protein, Bcl2, insidecells does not inhibit bispecific antibody efficacy as judged byequivalent luminesce decrease to control condition, confirmingnon-cytopathic efficacy of antibody action, along with a novel potentialsafety feature to further prevent hepatocyte death (FIG. 15 ).

Example 3 In-Situ Liver Expression of HBsAg/CD3-Bispecific Antibodiesfor HBV Immunotherapy

Hepatitis B virus (HBV) is a partially double stranded DNA virus withtropism to the liver, infecting over 300 million people chronicallyworldwide, causing cirrhosis and liver cancer in a significant number ofthese patients (E1-Serag, et al., 2012). Once infected, very few HBVpatients develop antibodies against and clear hepatitis B surfaceantigen (HBsAg), which serves a clinical biomarker for functional cure(Liu, et al., 2010). There is no effective treatment for chronic HBVpatients; a five year treatment course with entecavir, a reversetranscriptase inhibitor, results in HBsAg seroconversion in only 1.4% ofpatients (Chang, et al., 2010). These antiviral inhibitors suppressserum HBV DNA levels, but have no effect on covalently closed circularDNA (cccDNA), the episomal transcriptional template of HBV. Thismolecule is very stable once formed in the hepatocyte, and cccDNA hasbeen shown to persist for years (Werle-Lapostolle, et al., 2004).Pegylated interferon (IFN)-a is also approved for HBV therapy, but hasshown efficacy only in a minority of patients, while also being not welltolerated (Perrillo, et al., 2009).

In patients who clear HBV during the acute infection, the CD8-positiveT-cell response is crucial (Thimme, et al., 2003). This immune responseis, in part, noncytopathic, relying primarily on secreted cytokines,IFN-γ and tumor necrosis factor (TNF)-a, to mediate cccDNA degradation(Xia, et al., 2016). However, the frequency of HBV-specific T cells islow in chronically infected HBV patients (Boni, et al., 2007), and theirfunctionality is impaired (Park, et al., 2016). Given the paucity ofantiviral T cells in the host, T cells have been redirected to attackHBV-infected hepatocytes using chimeric antigen receptors (CAR) specificfor HBsAg (Bohne, et al., 2008). Redirected T cells were shown to reducecccDNA from infected primary hepatocytes in vitro (Bohne, et al., 2008),and mediate transient viral reduction in an HBV transgenic mouse model(Krebs, et al., 2013). While CAR-T cells represent a potential therapyagainst HBV, T-cell products currently have to be produced for eachpatient individually, limiting their potential utility as a readilyavailable therapeutic. To develop an “off-the-shelf product” to redirectT cells to HBsAg-positive hepatocytes, the inventors investigated herethe use of bispecific antibodies that recognize HBsAg and CD3, which isexpressed on almost all T cells.

Bispecific antibodies (Abs) targeting CD3 to direct T cells to cellsurface antigens were originally reported over 30 years ago (Staerz, etal., 1985; Staerz, et al., 1986)), and have shown promising antitumoractivity in numerous preclinical models. However, only blinatumomab, abispecific Ab that targets CD3 and CD 19, expressed on B-cellmalignancies, has received FDA approval so far (Przepiorka, et al.,2015). Current bispecific Ab approaches are challenged by a complicatedmanufacturing process, short half lives requiring continuous infusions,and side effects secondary to systemic T-cell activation (Mau-Sorensen,et al., 2015). These hurdles could be overcome through in situexpression of bispecific Abs from DNA or RNA templates in patienttissues, but there have been few reports on such strategies (Compte, etal., 2013; Pang, et al., 2017; Stadler, et al., 2017). The liver absorbsmajor fractions of gene therapy vectors, nanoparticles or liposomesallowing gene constructs to be delivered more readily than in any otherorgan. Expression of bispecific Abs in the liver should have severaladvantages compared to the passive infusion of recombinant proteins fortreating HBV. Local expression should result in increased Abconcentrations in the liver, before being diluted in the circulation.Moreover, soluble HBsAg in the serum of HBV patients can reduce efficacyby neutralizing a substantial fraction of infused Abs (Galun, et al.,2002), and the formed HBsAg/Ab immune complexes carry the risk ofimmune-complex disorders in HBV patients (van Nunen, et al., 2001).

To overcome these limitations, the inventors have developed an approachto express in situ a bispecific Ab to redirect T cells to HBsAg. THeresults in transfection-based murine models of HBV indicate a rapidreduction of the virus in a predominately noncytopathic manner.

Examples of Results

Hydrodynamic tail vein injection of a plasmid expressing HBsAg-specificantibodies results in the production of functional antibody in vivo. Toevaluate the feasibility of expressing functional HBsAg-specific Ab invivo, a minigene was cloned encoding a HBsAg-specific Ab (HBs-Fc),consisting of the immunoglobulin heavy-chain leader peptide, a singlechain variable fragment (scFv) derived from the HBsAg-specific Ab19.79.5 (Galun, et al., 2002; Eren, et al., 1998; Eren, et al., 2000),and the Fc domain of human IgG1, into the expression plasmid pCAG(pCAG.FIBs-Fc; FIG. 16A). Hydrodynamic tail vein (HTV) injection wasemployed to deliver plasmids into the liver, wherein a large volumebolus (10% fluid-body volume) is injected with plasmid DNA (Liu, et al.,1999), resulting in specific delivery into hepatocytes by punching holesinto cell membranes (Zhang, et al., 2004). Five or 15 μg of pCAG.HBs-Fcwas injected via HTV injection into immune competent mice, and theplasma concentration of HBs-Fc was measured 4 days post injection byELISA. Mean HBs-Fc concentrations were 6.4 mlU/mL for 5 μg and 14.7mlU/mL for 15 μg injected plasmid (FIG. 16B). Thus, HTV injection ofpCAG.HBs-Fc results in significant, dose-dependent production ofHBsAg-specific Abs in vivo.

HBs-Fc gene delivery has antiviral activity in vivo. To evaluate if invivo expression of HBs-Fc has antiviral activity a murine model wasadapted that allows for measuring the clearance of HBV usingnon-invasive bioluminescence imaging, which correlated with serum HBsAgand HBV DNA levels (Liang, et al., 2013). Bioluminescence of a reportergene had previously been shown to be a sensitive readout for CD8 T cellresponses in the liver against a co-delivered antigen gene (Stabenow, etal., 2010; Rai, et al., 2012). Briefly, a plasmid was generated encodingthe HBV genome and a green fluorescent protein (GFP)-2A-fireflyluciferase (GFP-2A-ffLuc) expression cassette, both under thetranscriptional control of identical HBV core promoters (pHBV-ffLuc;FIG. 21A). HTV injection of pHBV-ffLuc into NSG mice resulted inluciferase expression in the liver as judged by bioluminescence imaging,confirming the functionality of pHBV-ffLuc (FIGS. 21B,21C). Theintroduction of HBV plasmid DNA by HTV injection into immunocompetentmice results in immune clearance over two weeks, in a process resemblingacute HBV infection (Yang, et al., 2002). To evaluate if expression ofHBs-Fc induces clearance of HBV, pHBV-ffLuc was co-injected withpCAG.HBs-Fc, a pCAG plasmid encoding an Ab specific for an irrelevantantigen (Morgan, et al., 2012) (EGFRvIII; pCAG.EvIII-Fc), or a controlplasmid. Keeping the total amount of DNA injected consistent,co-injection of 5 μg pHBV-ffLuc with 15 μg pCAG.HBs-Fc resulted in asignificantly lower luciferase signal in comparison to co-injection with15 μg pCAG.EvIII-Fc or control plasmid (FIG. 17A). In addition,pHBV-ffLuc/pCAG.HBs-Fc co-injected mice had significantly lower levelsof serum HBsAg levels (FIG. 17B) in comparison to the other treatmentgroups, indicating that HBs-Fc has antiviral activity in vivo.

Including an anti-CD3 domain in HBs-Fc enhances the antiviral activityin vivo. Having established that pCAG.HBs-Fc has anti-HBV activity invivo, it was next determined if inclusion of a scFv specific for murineCD3, which activates T cells, further enhances its antiviral activity.pCAG expression plasmids were generated encoding HBs-Fc/CD3 orEvIII-Fc/CD3 bispecific Abs by inserting the murine CD3-specific scFvfrom mAb 145-2C11 (Leo, et al., 1987; Jost, et al., 1996) at thec-terminus of HBs-Fc or EvIII-Fc respectively (pCAG.HBs-Fc-CD3;pCAG.EvIII-Fc-CD3; FIG. 18A).

Keeping the total amount of DNA injected consistent, 5 μg pHBV-ffLuc wasinjected by HTV injection in combination with 15 μg control plasmid orplasmids encoding the respective Ab. In this study, pCAG.HBs-Fc,pCAG.HBs-Fc-CD3, EvIII-Fc, and pCAG.EvIII-Fc-CD3 were compared.Inclusion of a CD3-specific scFv enhanced the antiviral activity ofpCAG.HBs-Fc 30-fold (p<0.05) as judged by bioluminescence imaging (FIG.18B). Representative bioluminescence images of mice at day 4post-injection are shown, and at this time point there was a significantdifference between pCAG.EvIII-Fc and pCAG.EvIII-Fc-CD3 (p<0.05),indicating that bispecific Abs induce unspecific T-cell activation (FIG.18C). Unspecific T-cell activation was confirmed with a pCAG plasmidencoding an Ab specific for the irrelevant antigen EphA2 and CD3(pCAG.EphA2-Fc-CD3; (FIG. 22 ) (Iwahori, et al., 2015).

To evaluate the contribution of Fc receptor-mediated phagocytosis orcell killing to the observed antiviral activity of the bispecific Abs,the inventors replaced the wild-type human IgG1 Fc domain in HBs-Fc-CD3and EvIII-Fc-CD3 with a mutated human IgG4 Fc (mFc) domain that does notbind to Fc receptors (Hudecek, et al., 2015) (HBs-mFc-CD3;EvIII-mFc-CD3) (FIG. 19A). The antiviral activity was compared ofpCAG.HBs-mFc-CD3 to pCAG.EvIII-mFc-CD3 in the model. Both bispecific Abshad antiviral activity as judged by bioluminescence imaging (FIG. 19B).HBs-mFc-CD3 had significantly greater antiviral activity at day 4(10-fold) than EvIII-mFc-CD3 (FIG. 19B), while as shown previously usingthe non-mutated Fc (FIG. 18B), the HBs-Fc-CD3 had only 5-fold greateractivity than the corresponding EvIII-Fc-CD3 at the same time point.This observation was confirmed with additional replicates (FIG. 23 ),and therefore, HBs-mFc-CD3 and EvIII-mFc-CD3 were selected as a controlfor subsequent studies.

Bispecific antibodies act early after injection and in a CD3-dependentmanner in the HBV model. The inventors sought to explore the kinetics ofHBs-mFc-CD3 action over the course of acute clearance. The inventorsco-injected 5 μg pHBV.ffLuc and 15 μg pCAG.HBs-mFc-CD3 or Control,measuring bioluminescence each day for the first 4 days. There was a13-fold difference between control and HBs-mFc-CD3 treatment alreadyoccurs at day 1 post injection (FIG. 24A), with a similar differencemaintained subsequently. This matches the published kinetics of geneexpression HTV injection, which peaks at 8 hours post injection (Liu, etal., 1999), and suggests ongoing bispecific antibody production and/orantiviral T cell activation after day 1 is minimal.

The requirements were investigated for T cell signaling by substitutinga different moiety for T cell stimulation, replacing the CD3 targetingportion. For this purpose, the inventors utilized the extracellulardomain of the mouse CD80 protein, which can interact with CD28 expressedon T cells. This portion was cloned at the N-terminus in order to mostclosely resemble its natural orientation. The injectedpCAG.CD80-mFc-FIBs plasmid had significantly higher bioluminescence(28-fold higher) compared to pCAG.FIBs-mFc-CD3 plasmid (FIG. 24B),indicating that CD3 activation of T cells is critical for the observedantiviral activity.

In vivo expression of HBs-mFc-CD3 does not result in hepatocytetoxicity. Having demonstrated that unspecific T-cell activationcontributes to the antiviral activity of HBs-mFc-CD3, unspecifichepatotoxicity triggered by HBs-mFc-CD3 expression was explored. Theinventors first evaluated liver transaminase elevation that might resultfrom bispecific antibody expression, knowing the HTV procedure itselfresults in transient hepatocyte injury and serum alanineaminotransferase (ALT) and aspartate aminotransferase (AST) increases(Bonamassa, et al., 2011). Measured at day 4, no difference between theconditions were observed, with levels in AST still mildly elevated atthis time point post-injection (FIG. 25A). Since it is difficult toseparate bulk damage from the HTV procedure versus direct transgenetoxicity by examining transaminases alone, the inventors sought to moreprecisely study toxicity in individual transgene-modified hepatocytes.Toward this goal, a new hepatotoxicity assay was developed based onbioluminescence imaging to evaluate whether transfected hepatocytespersisted. Transgenic Rosa-Luc mice were injected by HTV injection withpCMV-NLS-Cre and pCAG.HBs-mFc-CD3 or control plasmids. The injectionresults in co-delivery of plasmids to the same hepatocytes; expressedCre recombinase in transfected cells of Rosa-Luc mice induces ffLucexpression, and the resulting bioluminescence signal correlates with thenumber of viable, transfected hepatocytes in vivo (details in methods).HBs-mFc-CD3 expression did not reduce the bioluminescence signal on day4 or 8 post injection versus control mice (FIG. 25B), indicating thatHBs-mFc-CD3 is non-toxic in hepatocytes. These findings were confirmedusing standard histological examination (H&E staining) of liver sectionsfrom mice co-injected with pHBV-ffLuc and control or pCAG.HBs-mFc-CD3 onday 4 post injection (FIG. 25C). Likewise, no histomorphological changesindicating toxicity were noted when transfecting previously used Abconstructs (FIG. 26 ). These histological stains also did notdemonstrate notable increase in lymphocyte levels among the differenttest constructs at day 4, indicating that additional T cells were notrecruiting into the liver, but rather tissue-resident T cells in theliver were likely activated (FIG. 26 ).

In vivo expression of HBs-mFc-CD3 has antiviral activity in arecombinant cccDNA HBV mouse model. Finally, the inventors wanted toexplore the ability of HBs-mFc-CD3 to induce cccDNA clearance in vivo,which more closely mimics HBV transcriptional templates in human cellsthan plasmids carrying the HBV genome. The inventors adapted previouslyreported HBV murine models that utilize recombinases to generate arecombinant cccDNA-like (rcccDNA) molecule lacking bacterial DNA (Qi, etal., 2014; Guo, et al., 2016). In the system, a floxed HBV genome wasconstructed with an LS-Cre recombinase (containing an internal intron)cassette driven by a CMV promoter in cis on the same plasmid (pCLX; FIG.27A; Kruse R L, et al, manuscript in preparation). In the floxed,unexcised state with Cre recombinase, there is no detection of HBVantigens, since viral transcripts and/or proteins are interrupted by theLoxP sequences preventing expression (FIGS. 27B,27C). After HTVinjection of pCLX, Cre expression and resultant rcccDNA formation yieldshigh level of HBsAg production and HBV core expression one week postinjection, demonstrating the functionality of the model (FIGS. 27B,27C).When pCLX is introduced into Rosa-Luc mice, the Cre recombinase alsoinduces ffLuc expression. Thus, bioluminescence imaging serves as anoninvasive means to monitor viable transfected cells and, as inprevious Rosa-Luc experiments, can be used to monitor hepatotoxicity ofthe bispecific Ab therapy.

To determine the antiviral activity and safety of HBs-mFc-CD3 in thercccDNA model, the inventors co-injected pCLX with pCAG.HBs-mFc-CD3,pCAG.EvIII-mFc-CD3 or control plasmid. On day 4 post injection 495-foldand 30-fold lower HBsAg levels were measured in pCAG.HBs-mFc-CD3injected mice compared to mice receiving control plasmid orpCAG.EvIII-mFc-CD3 respectively (FIG. 20A). pCAG.HBs-mFc-CD3 therapy notonly reduced HBsAg levels, but also induced HBsAg Abs earlier and athigher levels than p.CAG.EvIII-mFc-CD3 and controls (FIG. 20B). Theantiviral activity of pCAG.HBs-mFc-CD3 therapy was also confirmed byimmunofluorescence in a subset of animals on day 4 post injection,showing less HBV core expression within individual hepatocytes comparedto pCAG.EvIII-mFc-CD3 and control groups (FIG. 20C). Bioluminescenceimaging was used to determine the safety of pCAG.HBs-mFc-CD3 therapy. Onday 4 post injection there was 3.3-fold reduction in bioluminescencesignal in pCAG.HBs-mFc-CD3 injected mice in comparison to mice receivingcontrol plasmid (FIG. 20D), and long-term follow up revealed a furtherdecline of bioluminescence signal of pCAG.FIBs-mFc-CD3-injected mice. Atday 4, 20, and 26 post injection, there were significant differencesbetween pCAG.FIBs-mFc-CD3- and pCAG.EvIII-mFc-CD3-injected mice (p<0.05,FIG. 20E).

Significance of Certain Embodiments

Provided herein is a novel therapeutic strategy using bispecific Abs forHBV immunotherapy. Described herein is the production of bispecific Absin the liver leading to local T-cell activation, modulating the immuneresponse in the organ. This differs from recombinant protein strategies,which do not specifically accumulate at tissue sites unless additionaltargeting moieties are included, or from using cell-based carriers fordelivery (Iwahori, et al., 2015). The strategy utilizes the recruitmentof naive T cells against HBV, as opposed to relying on exhausted,dysfunctional HBV-specific T cells (Boni, et al., 2007; Park, et al.,2016) to mediate antiviral effects.

As shown herein, Abs can be expressed in hepatocytes and retain theirfunctionality. As expected, the HBs-Fc Ab was specific and effectivelymediated an antiviral effect, similar to the parent monoclonal Ab(Galum, et al., 2002; Eren, et al., 2000), while the addition ofCD3-specific scFv led to an even more potent antiviral response. Thisantiviral effect peaked at day 1 post-injection, and the inventors wereunable to detect significant levels of bispecific antibodies in theserum at day 4 likely due to activation and/or engagement with mouse Tcells preventing high-level accumulation. Delivering co-stimulationthrough CD80 ectodomain instead of CD3-specific scFv did not elicithigh-level antiviral responses. Interestingly, the bispecific Abtargeting CD3 and an unrelated antigen had significant anti-HBV activityin vivo, indicating that T-cell activation occurs independent of HBsAgbinding. Fc receptor cross-linking did not play a role in enhancingantiviral effects or in facilitating T-cell activation (Rinnooy, et al.,1986), since the construct with mutated Fc receptor binding sites,HBs-mFc-CD3, had similar anti-HBV activity to HBs-Fc-CD3.Antigen-independent secretion of IFN-γ has been observed previously invitro with the bispecific Ab format used in this study (Kuo, et al.,2012). In addition, antigen-independent T-cell activation by bispecificAbs secreted from cell lines has also been reported (Compte, et al.,2014). In specific embodiments, while not being bound by theory, themechanism of antigen-independent T-cell activation by bispecific Absoccurs through self-aggregation, resulting in crosslinking of CD3.

In this disclosure, a novel procedure was utilized for dual monitoringof hepatocyte viability and HBV markers, adapting recombination methodsto generate cccDNA-like molecules similar to previous reports (Qi, etal., 2014; Guo, et al., 2016), while also leveraging recombination ofhost cell reporter genes. The study was able to replicate other reportsthat initial T-cell effects against HBV in the liver are noncytopathic(Xia, et al., 2016), since the HBsAg levels were reduced to a muchlarger extent in the bispecific Ab treated groups compared to radiance,reflecting hepatocyte viability. Beyond activating T cells, HBs-mFc-CD3may also have reduced serum HBsAg levels in the rcccDNA model via theability of Abs to block HBsAg secretion from hepatocytes through theirengagement with neonatal Fc receptor (Neumann, et al., 2010). A laterdecrease in radiance in groups treated with bispecific Abs was observed,similar to the pattern of final clearance of infected cells inchimpanzees (Thimme, et al., 2003). In conjunction with the higherlevels of HBsAg IgG Ab production, this indicates bispecific Absincreased the host adaptive immune response versus control group.

The study differs from previous gene therapy studies demonstrating thatexpressing an individual cytokine, IFN-γ, in the liver has antiviralactivity (Dumortier, et al., 2005; Shin, et al., 2005). When tested in asurrogate woodchuck model, adenoviral delivery of IFN-γ and TNF-α didnot result in immediate reduction in the woodchuck hepatitis virus(WHV), but rather the later adaptive response against adenovirus by Tcells decreased WHV viral loads, suggesting that expressing individualcytokines alone may not be sufficient (Zhu, et al., 2004). Thusactivating T cells through CD3, which results in the production of notonly IFN-γ, but also other proinflammatory cytokines such as TNF-α andGM-CSF (Sen, et al., 2001), might be more effective. The targetedapproach to HBV is also distinct from the infusion of a recombinantTCR-like Ab to deliver IFN-α to HBV-infected hepatocytes (Ji, et al.,2012), and gene therapy with a Apo-Al/IFN-α fusion protein (Berraondo,et al., 2015). In addition, the strategy targeting HBsAg and CD3 formatdiffers from previous protein-based bispecific antibody strategiestargeting two different epitopes on HBsAg (Park, et al., 2000; Tan, etal., 2013), as well as a strategy targeting HBx and CD3 forhepatocellular carcinoma (Liao, et al., 1996).

In some embodiments, one can employ clinical translatable liver genedelivery systems, such as AAV (Nathwani, et al., 2011) ormRNA-nanoparticles (Thess, et al., 2015; DeRosa, et al., 2016). In arecent study, mRNA-nanoparticles effectively targeted liver in mice andsystemically secreted bispecific antibodies to engage claudin-6 and CD3triggering T-cell cytotoxicity against subcutaneously injected tumors(Stadler et al., 2017). This strategy is applicable to methods herein totreat in situ liver disease, in specific embodiments. Another limitationis that HTV injection results in an acute model of HBV, rather than achronic HBV mouse model, which can be established in immunocompetentmice with AAV (Dion, et al., 2013) or low-dose adenoviral (Huang, etal., 2012) delivery of HBV genomes.

In conclusion, a novel therapeutic approach to target HBV infection byexpressing bispecific Abs in hepatocytes, leading to a local andpredominantly noncytopathic immune response against HBV, is providedherein. THe approach is of value not only for treating HBV but alsoother viral liver diseases.

Examples of Materials and Methods

Plasmid constructs. Generation of Ab constructs: A codon-optimizedminigene was synthesized by IDTDNA (Coralville, IA) containing theimmunoglobulin heavy-chain leader peptide (MDWIWRILFL VGAATGAHS; SEQ IDNO.), the HBs-specific mAb 19.79.5 (Eren, et al., 1998) heavy-chain, aglycine (G) serine (S) linker [(G45)3], and the 19.79.5 light-chainflanked by 5′ Xhol and 3′ BamHI sites. The human IgG1 Fc domain was PCRamplified from a plasmid encoding a CAR containing a IgG1 Fc hinge withthe PCR primers containing 5′ BamHI and NotI 3′. The minigene and PCRproduct was cloned into pCAG by three-way ligation to createpCAG-HBs-Fc. To create the EvIII-Fc control plasmid, the 139 scFvspecific for EGFRvIII was PCR cloned from pSFG.139-CD3-I-mOrange withPCR primers containing 5′ Xhol and 3′ BamHI sites. Three-way ligationreaction was performed to create pCAG-EvIII-Fc. Cloning was confirmed bysequencing (Lone Star Labs, Houston, TX).

Generation of bispecific Ab constructs: The 145-2C11 (Leo, et al., 1987)scFv specific for murine CD3 was PCR amplified frompRV2011.145-2C11-1D3-I-Thy1.1 with 5′ EcoRV and 3′ NotI sites. Four-wayligations was performed with 5′ Xhol-leader-HBs or EvIII scFv-3′ BamHI,5′ BamHI-Fc-3′ EcoRV, 5′ EcoRV-145-2C1 1 scFv-3′ NotI, and pCAG digestedwith Xhol and NotI to generate pCAG.HBs-Fc-CD3 and pCAG.EvIII-Fc-CD3.pCAG.HBs-mFc-CD3 and pCAG.EvIII-mFc-CD3 were generated in a similarfashion except a codon optimized minigene (IDTDNA, Coralville, IA)encoding the human IgG4 Fc with mutated Fc binding sites (Hudecek, etal., 2015) and flanking 5′ BamHI and 3′ EcoRV sites was used instead of5′ BamHI-Fc-3′ EcoRV. pCAG.CD80-mFc-HBs was generated by synthesizingthe extracellular domain including leader sequence of murine CD80 (B7.1)protein between 5′-Xhol and 3′ BamHI sites. Separately, PCRamplification of HBs scFv added 5′ EcoRV and 3′ NotI sites. Four-wayligation reaction with 5′-XhoI-CD80-3′ BamHI, 5′ BamHI-mFc-3′ EcoRV, and5′ EcoRV-HBs-3′ NotI, and pCAG digested with Xhol and NotI was performedto generate pCAG.CD80-mFc-HBs. Cloning was confirmed by sequencing (LoneStar Labs, Houston, TX).

Generation of pCAG: The pCAG vector was constructed from the pCIG vector(containing the hybrid promoter CMV enhancer chicken beta actin (CAG)promoter, rabbit beta globin 3′UTR, polyadenylation sequence,IRES-LS-GFP, and SV40 origin of replication) by removing IRES-NLS-GFPleaving Xhol and NotI sites for inserting transgenes (FIG. 21A).

Generation of pHBV-ffLuc: pHBV-ffLuc was generated by inserting aGFP-2A-ffLuc expression cassette by PCR cloning (provided by Dr. InderVerma, Salk Institute, San Diego, CA) into the Smal and Sacl sites ofpSP65ayw1.3 (provided by Dr. Stefan Wieland, University of Basel,Switzerland). pSP65ayw1.3 encodes an over-length HBV genome fromgenotype D, subtype ayw, GenBank V01460. The Smal site is located at the3′-end of the over-length HBV genome and the PCR primers forGFP-2A-ffLuc subcloning were designed so that GFP-2A-ffLuc translationis in frame with core protein translation at the 3′ end of the HBVgenome (see FIG. 28 for more sequence information). Cloning wasconfirmed by sequencing (Lone Star Labs, Houston, TX).

Bioluminescence imaging. The IVIS® system (Xenogen Corp., Alameda, CA)was used for bioluminescence imaging. Mice were anesthetized withisofluorane and injected intraperitoneal with 200 μE of 7.5 mg/mLluciferin solution (GoldBio, Olivette, MO). Luciferin was allowed tocirculate for 10 minutes post-injection, and mice were placed ventralside up and imaged promptly thereafter. Luminescence signals werequantified using Living Image 4.2 software (Caliper Life Sciences,Hopkinton, MA) with a region of interest (ROI) circling the area overthe liver.

HBsAg ELISA. HBsAg levels were determined as previously described(Billioud, et al., 2016). Briefly, serum HBsAg levels were evaluatedwith commercially sold ELISA kits according to manufacturer'sinstructions (International Immuno Diagnostics, Foster City, CA).Quantification of serum HBsAg was made by comparing serial dilutions ofknown standards (Alpha Diagnostic International, San Antonio, TX). HBsAglevels were reported as ng/mL, consistent with the standards utilized.The conversion ratio to IU/mL is not provided by the manufacturer, butmany kits have conversions of 1 or 10 ng/mL to 1 IU/mL HBsAg asapproximate guidelines (Locarnini, et al., 2012).

HBsAg IgG antibody ELISA. Serum HBsAg IgG Ab levels were quantified byELISA according to manufacturer's instructions (Alpha DiagnosticInternational, San Antonio, TX). HBsAg IgG Ab levels were reported asmlU/mL. The ELISA assay can detect both human and mouse immunoglobulin.

Transaminase analysis. Serum alanine aminotransferase (ALT) andaspartate aminotransferase (AST) were measured by the ComparativePathology Laboratory (Baylor College of Medicine, Houston, TX) usingCOBAS INTEGRA 400 plus analyzer (Roche Diagnostics, Indianapolis, IN).

Animal Experiments. All animal experiments followed a protocol approvedby the Baylor College of Medicine Institutional Animal Care and UseCommittee. For all experiments using immunocompetent mice, the Rosa-Lucstrain from Jackson Labs (FVB.12956(B6)-Gt(ROSA)26Sortml(Luc)Kael/J) wasutilized in which the expression of the firefly luciferase (Luc) gene isblocked by a loxP-flanked STOP fragment placed between the Luc sequenceand the Gt(ROSA)26Sor promoter. Even though most experiments did notutilize the endogenous luciferase reporter, using Rosa-Luc mice for allexperiments reduced the risk of inter-strain variability. Rosa-Luc mice,aged 6-10 weeks, were selected for hydrodynamic tail vein injection. Asa filler or control plasmid so that equal amount of DNA was injected ineach group of mice, pCMV-Gaussia luciferase (Therm oFisher, Waltham, MA)was used. Plasmid DNA was diluted into 0.9% normal saline solution to atotal volume equaling 10% of murine body weight. Mice were placed undera heat lamp for 5-10 minutes to dilate the lateral tail veins, andinjection performed over 4-6 seconds (Kovacsics, et al., 2014). Micewere bled retro-orbitally, and serum collected after centrifugation for30 minutes at 2.3 G. Serum was stored at −80»C until further use forHBsAg ELISA and HBsAg IgG ELISA quantification.

Histology. Frozen tissue slides from livers were fixed with 4% PFA for10 minutes, and stained for HBV core overnight at 4° C. in PBS-T buffer(PBS IX containing 0.5% BSA and 0.2% of triton-100) using the primaryantibody: rabbit anti-hepatitis B virus core antigen (Dako/Agilent,Santa Clara, CA). Primary antibody was washed with PBS IX, and slideswere incubated with Alexa-Fluor secondary antibodies (Molecular Probes,Eugune, OR) in PBS− T buffer. Vectashield plus DAPI (Vector Labs,Burlingame, CA) was used for slides mounting. In other experiments,liver tissue was fixed in 4% paraformaldehyde overnight, and serialsections of paraffin-embedded liver stained with hematoxylin & eosin.

Statistical analysis. Statistical analysis was performed using GraphPadPrism 7 software (GraphPad Software, Inc., La Jolla, CA). Datameasurements are presented as mean+/−standard error of mean (s.e.m.).Mean differences were tested using appropriate tests including unpaired,parametric, one-tailed t-tests. Significance level used was p<0.05,unless otherwise specified.

REFERENCES

All patents and publications mentioned in the specification areindicative of the level of those skilled in the art to which thedisclosure pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

PATENTS AND PATENT APPLICATIONS

-   US 2002/0151509-   US2011/0184049

PUBLICATIONS

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Although the present disclosure and its advantages have been describedin detail, it should be understood that various changes, substitutionsand alterations can be made herein without departing from the spirit andscope of the disclosure as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present disclosure, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present disclosure.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

What is claimed is:
 1. A method of producing an antibody againsthepatitis B virus (HBV) in situ in a tissue within a subject, comprisingthe steps of: a) administering two or more polynucleotides directly tothe tissue within the subject, wherein each of the two or morepolynucleotides each encoding non-identical monospecific antibodypolypeptides; and b) generating antibodies in situ in the tissue withinthe subject, wherein the generated antibodies dimerize to each other toproduce a mixture of monospecific antibody or bispecific antibodypolypeptides against HBV.
 2. The method of claim 1, wherein themonospecific antibody or the bispecific antibody comprises one or moreantigen-binding domains, wherein each of the antigen-bending domainscomprising a single chain antibody, a single chain variable fragment(scFv), peptide, camelid variable domain, shark IgNAR variable domain,single domain antibody, affimer or VHH antibody.
 3. The method of claim1, wherein the polynucleotides further encode at least one liverantigen-targeting entity and/or immunostimulatory entity.
 4. The methodof claim 3, wherein the liver antigen-targeting entity comprises asingle chain antibody, a single chain variable fragment, a camelidantibody, or a peptide.
 5. The method of claim 3, wherein theimmunostimulatory entity comprises an anti-CD3 scFv, an anti-CD28 scFv,anti-41BB scFv, anti-OX40 scFv, anti-CTLA4 scFv, anti-CD16 scFv,anti-PD1 scFv, anti-PD-L1 scFv, anti-CD47 scFv, part or all of theectodomain for a ligand for CD28, part or all of the ectodomain of 41BBligand, part or all of the ectodomain of the LIGHT protein, ICOS-ligand,CD276 (B7-H3), B7-H4, B7-H6, CD134L, CD137L, or a cytokine.
 6. Themethod of claim 1, wherein the polynucleotides are included in a vector.7. The method of claim 6, wherein the vector is adeno-associated virus(AAV), wherein the vector comprises a tissue-specific promoter.
 8. Themethod of claim 1, wherein the antibody against (HBV) is specific to HBVsmall surface antigen, HBV middle surface antigen, HBV large surfaceantigen.
 9. A method of producing a bispecific, trispecific orquadraspecific antibodies in situ in a tissue within a subject,comprising the steps of: a) administering two or more polynucleotidesdirectly to the tissue within the subject, wherein each of said two ormore polynucleotides encodes non-identical bispecific antibodypolypeptides, wherein the antibodies produced from the polynucleotidesin situ in the tissue of the subject dimerize to each other, and b)producing a mixture of bispecific, trispecific or quadraspecificantibodies in situ in the tissue within the subject.
 10. A method oftreating hepatitis B in a subject having HBV infection, comprising thesteps of: a) administering a therapeutically effective amount of avector directly to the tissue within the subject, wherein the vectorcomprising two or more polynucleotides each encoding non-identicalmonospecific antibody polypeptides; b) generating antibodies in situ inthe tissue within the subject, wherein the non-identical monospecificantibody polypeptides dimerize to each other to produce a mixture ofbispecific antibody polypeptides against HBV and havingimmunostimulating capacity; and c) reducing the HBV viral load withinthe tissue.
 11. The method of claim 10, wherein the bispecific antibodycomprises one or more antigen-binding domains, wherein each of theantigen-bending domains comprising a single chain antibody, a singlechain variable fragment (scFv), peptide, camelid variable domain, sharkIgNAR variable domain, single domain antibody, affimer or VHH antibody.12. The method of claim 10, wherein the vector encodes at least oneliver antigen-targeting entity and/or immunostimulatory entity.
 13. Themethod of claim 12, wherein the liver antigen-targeting entity comprisesa single chain antibody, a single chain variable fragment, a camelidantibody, or a peptide.
 14. The method of claim 12, wherein theimmunostimulatory entity comprises an anti-CD3 scFv, an anti-CD28 scFv,anti-41BB scFv, anti-OX40 scFv, anti-CTLA4 scFv, anti-CD16 scFv,anti-PD1 scFv, anti-PD-L1 scFv, anti-CD47 scFv, part or all of theectodomain for a ligand for CD28, part or all of the ectodomain of 41BBligand, part or all of the ectodomain of the LIGHT protein, ICOS-ligand,CD276 (B7-H3), B7-H4, B7-H6, CD134L, CD137L, or a cytokine.